Antimicrobial Activity and Effectiveness of Dishwashing Liquid derived from

Citrus Microcarpa Fruit Peel Extract against Salmonella spp.

An Undergraduate Research

Presented to the

Faculty of the College of Allied Health Sciences

PHINMA Cagayan de Oro College

Cagayan de Oro City

In Partial Fulfillment

of the Requirements for the Course

Bachelor of Science in Medical Laboratory Science

Abang, Elmie Jane E.

Abriam, Kate Hyacinth

Alboradora, Aimei Pearl C.

Aljas, Kaye Angelie N.

Arellano, Kean Harvey E.

Barrita, Shawney M.

Balatayo, Louise Brylle O.

Balbutin, Joshua O.

Matbagan, Rosh Hashana Louisse S.

Naïve, Louilla Mae M.

March 2024

0
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Chapter 1

INTRODUCTION

Background of the Study

Nowadays, one of the significant problems in our community is getting diseases

from microorganisms acquired through kitchen utensils. Pathogens on spoons and forks

could cause acute and chronic health consequences with varying durations, severity, and

mortality, resulting in significant societal expenditures and burdens (Debuisson,

2021). Salmonella infection was concerning since it was a significant source of food-borne

illness (salmonellosis) and accounted for most instances of disease each year. A

dishwashing liquid derived from C. microcarpa fruit peel extract was said to be effective

as it had antioxidants and antibacterial activity (Afriyandi et al., 2020).

Bacteria and viruses, often known as germs, could dwell on surfaces such as table

tops, kitchen countertops, picnic tables, and desktops, and could spread into contact with

a person's hand and mouth (Debuisson et al., 2021). According to the Centers for Disease

Control and Prevention (CDC), approximately one in every six Americans (or 48 million

people) becomes ill each year, 128,000 are hospitalized, and 3,000 die as a result of food-

borne infections in 2011 (Debuisson et al., 2021). The presence of potentially harmful

bacteria such as Staphylococcus aureus, Salmonella, Shigella, and Vibrio species may

have been of public health concern. The requirement for kitchen disinfection and covering

kitchen utensils became a critical necessity that developing countries needed to address

in their policymaking (Aliyu et al., 2023).

C. microcarpa (calamondin), an evergreen tree native to the tropics and subtropics,

is known as "calamandarin" and China orange (Chen et al., 2023). It has been used as an
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immune booster for decades; its antioxidant and antibacterial effects led to the creation

and stimulation of the immune system in response to pathogens invading the body (Gamis

et al., 2023). Based on the literature, C. microcarpa fruit peel extract encompassed

flavonoids and a monoterpene hydrocarbon with bioactivity that are antimicrobial,

antibacterial, and antioxidant, which correlated to its bactericidal and antimicrobial

properties (Mahardika & Roanisca, 2020). According to the literature, the antibacterial

activity of alkaloids, phenols, and flavonoids caused damage to bacterial cell walls,

changed membrane permeability, inhibited enzyme activity, and disrupted protein

synthesis (Mahardika & Roanisca, 2020). The primary role of the dishwasher detergent is

to remove filth from dishes, tableware, kitchen equipment, and surfaces that come into

contact with food (Rajca & Wieczorek, 2021).

The purpose of the study was to unleash the importance of using C. microcarpa

fruit peel extract as an ingredient in making dishwashing liquid and to have an organic-

based dishwashing liquid that was effective in eliminating bacteria. The researchers

sought to address the potential for a natural, citrus-based solution to enhance kitchen

hygiene, combating the prevalence of Salmonella spp. contamination in household

settings. This study gave every household, cafeteria, and food vendor the benefit of using

dishwashing liquid that was accessible and not harmful.

The researchers tested the efficacy by conducting an experiment showing that the

extract could be used as a dishwashing liquid for every community, particularly in areas

prone to acquiring food-borne diseases. The collected data from the experiment was

analyzed and provided helpful knowledge to people who had difficulty eliminating all

bacteria in utensils that were not visible to the naked eye. It had an impact on choosing a

dishwashing liquid that could potentially eliminate bacteria and prevent the acquisition of

diseases.
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Theoretical Framework

This study anchored by the Germ theory by French chemist Louis Pasteur in the

1860s which explains how tiny organisms called germs could invade the body and that

microorganisms cause certain diseases and follow the asepsis technique to eliminate the

microbes causing disease (Debre, 2022). In which certain bacteria present in the kitchen

utensils are a significant cause of food-borne disease. Based on the principle of antibiotic

susceptibility testing used in microbiology laboratories, this was employed to identify the

precise antibiotics to which a particular bacterium or fungus is sensitive. The minimum

inhibitory concentration (MIC) is the lowest medication concentration that prevents an

organism from growing and is a typical way to describe results. Reports often include a

qualitative interpretation and a quantitative result in g/mL. Each result is usually assigned

one of the following interpretation categories: susceptible (S), intermediate (I), resistant

(R), sensitive-dose dependent (SD), or no explanation (NI) (Street, 2022).

The study correlated with the Germ Theory as it focused on disease transmission

through kitchen utensils that carried harmful bacteria, particularly Salmonella spp. The

study contributed to addressing this concern by exploring the effectiveness of natural,

citrus-based solutions in enhancing kitchen hygiene to mitigate the threat of getting

disease from dirty kitchen utensils. As such, the bioactive compound present in the C.

microcarpa was known for its antimicrobial properties, the different percent concentrations

of the dishwashing liquid were seen as a way to test the effectiveness of the product, and

the type of dishwashing liquid significantly eliminated bacteria causing disease. Overall,

the study aligned with the principles of Germ theory by focusing on the role of

microorganisms in disease transmission and the potential use of antimicrobial agents to

mitigate their impact.

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Conceptual Framework

The study reveals two variables employed in the study's execution. The independent

variables are the bioactive compound in dishwashing liquid derived from C. microcarpa

fruit peel extract (Flavonoid, Terpenoid, and Phenolic/Tannis), concentrations of C.

microcarpa fruit peel extract at different levels (50%, 70% & 100%) and the type of

dishwashing liquid (C. microcarpa fruit peel extract-based and commercial dishwashing

liquid). The dependent variable is mainly antimicrobial activity and effectiveness of

dishwashing liquid derived from C. microcarpa fruit peel extract against Salmonella spp.

(measured by zone a zone of inhibition, with ciprofloxacin as control). The extract from C.

microcarpa peel was tested and validated as the experiment was conducted. The

experiment produced an effective dishwashing liquid derived from C. microcarpa fruit peel

extract.

The relation between the independent and dependent variables was that changes in

the independent variables (such as the composition of bioactive compounds,

concentration levels, and type of dishwashing liquid) were expected to influence the

dependent variable (antimicrobial activity and effectiveness of dishwashing liquid derived

from C. microcarpa against Salmonella spp.). For instance, different concentrations of the

fruit peel extract or variations in the types and proportions of bioactive compounds could

lead to variations in the antimicrobial effectiveness of the dishwashing liquid.

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INDEPENDENT VARIABLE DEPENDENT VARIABLES

Bioactive compounds in dishwashing

liquid derived from C. microcarpa fruit
peel extract.
 Flavonoids
 Terpenoids
 Phenolic/Tannis

Antimicrobial Activity and

Concentrations of C. microcarpa fruit Effectiveness of dishwashing liquid
peel extract (at different levels) derived from C. microcarpa fruit peel
extract against Salmonella spp..
 50% (measured by zone of inhibition, with
 70% ciprofloxacin as control)
 100%

Type of dishwashing liquid

 C. microcarpa fruit peel
extract-based
 Commercial dishwashing
liquid.

Figure 1. Schematic Presentation Showing the Integrity between the Independent and
Dependent Variables of the study.

Statement of the Problem

In the industry, one of the major problems that we faced was all about cross-

contamination, foodborne diseases, and even health complications due to improper

disinfectant or cleaning materials in our kitchen utensils. Contaminated utensils could

harbor harmful bacteria like Salmonella spp., leading to foodborne illnesses when these

bacteria come into contact with food during preparation or consumption. In addition, dirty

kitchen utensils led to various health complications, ranging from mild gastrointestinal

discomfort to severe cases requiring medical intervention. In line with that, the need to
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evaluate the antimicrobial activity and effectiveness of dishwashing liquid derived from C.

microcarpa fruit peel extract was viewed as a possible alternative cleaning solution that

could inhibit the growth of the bacteria.

Objectives of the Study

This study aimed to investigate the antimicrobial properties of dishwashing liquid

derived from C. microcarpa fruit peel extract, specifically targeting its effectiveness against

Salmonella spp.

1. To identify the bioactive compounds present in the C. microcarpa fruit peel extract;

2. To measure the antimicrobial activity of dishwashing liquid derived from C.

microcarpa fruit peel extract at different concentrations (50%, 70%, and 100%)

against Salmonella spp. as measured through the zone of inhibitions;

3. To compare the effectiveness of dishwashing liquid derived from C. microcarpa

fruit peel extract in different concentrations, and commercial dishwashing liquid

against Salmonella spp.; and

4. To determine the bioactive compounds present in dishwashing liquid derived from

C. microcarpa fruit peel extract at different concentrations significantly influence

the antimicrobial activity and effectiveness of C. microcarpa fruit peel extract

compared to commercial dishwashing liquid against Salmonella spp.

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Hypothesis

Ho: The dishwashing liquid derived from C. microcarpa fruit peel extract is not

effective against Salmonella spp.

Ha: The dishwashing liquid derived from C. microcarpa fruit peel extract is effective

against Salmonella spp.

Significance of the Study

The study on the antimicrobial activity and effectiveness of dishwashing liquid

derived from C. microcarpa fruit peel extract against Salmonella spp. was vital because it

could provide valuable solutions on how to prevent foodborne diseases acquired from dirty

kitchen utensils. The data gathered from this study was crucial to the Department of

Health, Department of Agriculture, community, and future researchers who were advised

to expand their knowledge and awareness about factors such as dirty kitchen utensils that

result in foodborne diseases. The findings of this study benefited the Department of

Health, the Department of Agriculture, the community, and future researchers who wanted

to improve their knowledge and skills in making dishwashing liquid that could eliminate

pathogens.

Department of Health. The study provided valuable information to the Philippine

Health Agency about the antimicrobial activity and effectiveness of dishwashing liquid

derived from C. microcarpa fruit peel extract against Salmonella spp. The results enabled

the agency to set standard guidelines for washing kitchen utensils and formulate a more

effective dishwashing liquid to prevent the rising cases of foodborne diseases.

Department of Agriculture. The study provided an opportunity for the Philippine

agency to further cultivate locally farm-produced C. microcarpa and provided a

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multifaceted approach to produce dishwashing liquid that possessed antimicrobial activity

and effectiveness that could significantly inhibit the growth of bacteria.

Community. This research assisted those communities that played an essential

role in raising awareness about antimicrobial activity and the effectiveness of dishwashing

liquid derived from C. microcarpa fruit peel extract against Salmonella spp. It further

helped them understand the relevance and impact of dishwashing liquid with bioactive

compounds towards safer food consumption.

Future Researchers. This research was a valuable reference for researchers who

planned to conduct any related studies on the effectiveness of dishwashing liquid derived

from C. microcarpa fruit peel extract in kitchen utensils in response to bacteria-causing

diseases.

Scope and Limitations

Testing of the study was conducted at Central Mindanao University during the

given research time framework of the school year 2023-2024. In line with this study, the

researchers observed the efficacy of the dishwashing liquid derived from C. microcarpa

peel extract on Salmonella spp. based on its zone of inhibition in different concentrations.

The center of the study identified the effectiveness of dishwashing liquid derived

from C. microcarpa fruit peel extract on Salmonella spp. This study is limited to the

following: the peel of the plant, the remaining parts such as the juice, stalk, leaves, and

roots that are not used as the researcher seeks to find out the synergistic activity of the

peel alone; the formulation dishwashing liquid derived from C. microcarpa fruit peel extract

limits only in water-based; and specific bacteria, Salmonella spp. as it is the most isolated

bacteria in the kitchen settings.

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Definition of Terms

Antimicrobial. It is the ability of the dishwashing liquid containing citrus

microcarpa (C. microcarpa) fruit peel extract to inhibit the growth or kill Salmonella spp.

bacteria on kitchen utensils.

Aseptic technique. It refers to a set of procedures and practices done in the

formulation of the extract to prevent contamination.

Bacteria. It refers to microorganisms, specifically Salmonella spp., that could

potentially contaminate kitchen utensils and cause foodborne illnesses.

Bacterial Culture. It is a method of multiplying microbial organisms by letting them

reproduce in a predetermined culture medium under controlled laboratory conditions.

Citrus microcarpa. It contains antimicrobial activity and was used as the source

of peel in the study.

Citrus Essential Oil. It is the product when citrus peel is boiled in water. It contains

antimicrobial activity that can kill microorganisms like Salmonella spp.

Chelating agents. It is an ingredient used in dishwashing liquid formulation that

helps enhance the quality of the product by binding from the washing water.

Community. It refers to a group of individuals or households that used kitchen

utensils and were potentially exposed to Salmonella spp. contamination.

Detergents. It refers to chemical compounds used in the formulation of

dishwashing liquid that facilitated the removal of dirt, grease, and food residues from

kitchen utensils.
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Disinfection. It is a process that eliminates many or all pathogenic

microorganisms like Salmonella spp., the primary purpose of the product.

Diffusion method. It is a laboratory technique used to assess the antimicrobial

activity of the dishwashing liquid.

Dishwashing liquid. It is a liquid soap used to remove dirt and kill microbes from

kitchen utensils and was the main product in the study.

Distillation. It is the method employed in the study to produce essential oil from

the peel of the fruit.

Distilled water. It is the primary solvent used in the study in formulating extract

and dishwashing Liquid.

Domestic kitchens. It is the area where bacteria that cause infection grows.

Effectiveness. It refers to the ability of the dishwashing liquid to successfully

inhibit the growth or eliminate Salmonella spp. bacteria on kitchen utensils, thereby

reducing the risk of foodborne illnesses associated with bacterial contamination.

Essential Oil. It refers to the concentrated, volatile liquid extracted from the peel

of citrus microcarpa fruit.

Ethnobotanical. It involves investigating the historical and cultural significance of

C. microcarpa in traditional medicine or household applications, particularly its potential

antimicrobial properties when used in dishwashing liquid formulations.

Experiment. It is a systematic procedure conducted to investigate the

antimicrobial properties and effectiveness of the dishwashing liquid containing citrus

microcarpa fruit peel extract against Salmonella spp. bacteria on kitchen utensils.
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Extract Force. It is used to remove or squeeze the juice of the peel of C.

microcarpa.

Flavonoids. It is a biologically active compound that is derived from plants, which

gives plants their color, biological activity, and secondary antioxidant defense

mechanisms, which are abundant naturally occurring constituents.

Food-borne disease. It refers to illnesses caused by consuming contaminated

food, particularly those resulting from the presence of pathogenic bacteria like Salmonella

spp.

Gastroenteritis. It is also known as "stomach flu", which is inflammation of the

stomach and intestinal lining, which causes diarrhea and vomiting. The Salmonella spp.

could cause it.

Kitchen utensils. It is a small hand-held tool used for food preparation and the

major cause of food-borne disease.

Lemonsito. It is native to the Philippines and recognized by the Department of

Agriculture.

Minimum inhibitory concentration. It is the lowest concentration of an antibiotic

at which bacterial growth is completely inhibited, used in the study to measure the

effectiveness of the product.

Nutrient agar. Used to culture Salmonella spp.

Pathogens. It is a microorganism, such as Salmonella spp., that could cause

disease or illness in humans when ingested through contaminated food.

Pectin. It is a component in Calamansi skin as a natural fiber and is a plant-based

thickening agent.
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Peel. The outer protective layer of the C. microcarpa fruit could be peeled off and

was used to make essential oil.

Phenolic compounds. The natural bioactive molecules are found mainly in plant

tissues with interesting bioactivities, such as antioxidant, antimicrobial, anti-inflammatory,

and antiproliferative activities.

Phytochemical testing. It is the process of identifying the chemical compounds

produced by plants and testing was conducted at Central Mindanao University.

Resistant. It is a marker for an ineffective dishwashing liquid.

Rural Areas. It includes examining the use and effectiveness of the dishwashing

liquid in households located in rural areas, where access to commercial cleaning products

may be limited and where foodborne illness prevention is particularly important due to

potentially limited healthcare resources.

Rutaceae Family – It is a family of citrus plants, shrubs, and trees with tetramerous

or pentamerous flowers and a compound ovary with four or five discrete or partly joined

carpels.

Salmonellosis. Its symptoms ranged from normal gastroenteritis to enteric fevers,

and life-threatening infections requiring urgent and appropriate antibiotic treatment.

Salmonella spp.. It is a bacteria commonly found on kitchen surfaces and

dishware.

Sensitivity testing. It refers to the process of evaluating the susceptibility of

microorganisms, such as Salmonella spp., to the antimicrobial properties of the

dishwashing liquid.

Sponge. It is soft when wet, absorbs dishwashing liquid, and cleans dirty kitchen

utensils.
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Susceptible. It refers to the characteristic of microorganisms, such as Salmonella

spp., that were vulnerable or responsive to the antimicrobial effects of the dishwashing

liquid.

Symptoms. They are manifestations of disease acquired from Salmonella spp—

infection, with signs of disease perceived to include diarrhea and fever.

Terpenoids. They are volatile substances that give plants and flowers flavor and

fragrance, one of the bioactive compounds measured in the study.

Thickener. It is an ingredient added that increases the viscosity of dishwashing liquid.

Typhoid fever. It is also known as enteric fever, a potentially fatal multisystem

illness caused primarily by Salmonella spp.

Transmission. It is the process of passing something from one person, object, or

place to another through the transmission of kitchen utensils.

Zone of Inhibition Test. It is most frequently used to measure the sensitivity or

resistance of bacteria to known or potential antimicrobial agents.

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Chapter 2

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter presented studies that were inclusive of the existing research

concerning C. microcarpa properties, C. microcarpa peel, bioactive compounds,

dishwashing liquids and commercial dishwashing liquids, antimicrobial activity,

concentration, zones of inhibition, and Salmonella spp. The aim was to supplement the

research on the potential effectiveness of these components.

Citrus microcarpa

Citrus was a general term for several different orange species and hybrids; lemon

(Citrus lemon), lime (Citrus aurantifolia), sweet orange (Citrus sinensis), and, other citrus

fruits were of great commercial importance (Dachriyanus et al., 2021). Based on Tanaka's

classification system, citrus fruits belonged to the Rutaceae family, which included about

162 species and 160 genera, which comprised significant crops like oranges, lemons,

pummelos, grapefruits, limes, etc., that were distributed throughout the world (Alkatib et

al., 2022).

Citrus fruits, ranging from sweet, sour, bitter mandarins, lemons, limes, grapefruit,

pomelo, citrons, and many hybrids and cybrids, were members of the Rutaceae family

(Kabdal et al., 2023). Calamondin (C. microcarpa Bunge) was widely produced in

Indonesia and was even promoted by Calamondin One Village One Product (OVOP)

(Afriyandi et al., 2020). In 2019, the Food and Agriculture Organization of the United

Nations reported a global citrus production of 144 million tons, with China ranking first

globally in output and cultivation area (Gu et al., 2022).

Extracts of calamondin peel from three regions, namely Bukittinggi, Padang, and

Pesisir Selatan, were said to be effective as they had an antibacterial activity against
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bacteria Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and

Pseudomonas aeruginosa (Afriyandi et al., 2020). While the essential oil of calamansi peel

effectively inhibited methicillin-resistant Staphylococcus aureus, Pseudomonas

aeruginosa, Escherichia coli, and Staphylococcus aureus germs (Boukeskasse et al.,

2019). In contrast, the leaves of calamansi had extreme inhibitory power. Citrus is a plant

with numerous health advantages, and its pharmacological properties received much

attention (Dachriyanus et al., 2021). Citrus essential oil, which was abundant in limonoids

and terpenes and had powerful antioxidant, antibacterial, and flavorful characteristics, was

a crucial commodity for the economy (Gowda et al., 2022).

Citrus microcarpa peel

A study by Dachriyanus et al. (2021) demonstrated the antibacterial activity of

calamansi leaf essential oil. Tests conducted on the leaves demonstrated activity against

Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Aspergillus niger, Candida

albicans, and Saccharomyces cerevisiae. Meanwhile, the essential oil present in the fruit

peels was shown to be effective against Staphylococcus aureus, Pseudomonas

aeruginosa, Escherichia coli, and Enterococcus faecalis. The study aimed to identify the

significant compounds in the leaves and fruit peels responsible for their antibacterial

activity, particularly against resistant bacteria like MRSA (methicillin-resistant

Staphylococcus aureus), gram-positive bacteria (Staphylococcus aureus and

Streptococcus mutans), and gram-negative bacteria (Pseudomonas aeruginosa and

Escherichia coli). It would allow researchers to determine which essential oils possess the

most substantial antibacterial potential. As reported by Dai et al. (2019), essential oils can

exhibit bacteriostatic or bactericidal activity against infections, thus making them

potentially useful as natural antibacterial agents.

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Bioactive compound

Studies on the phytochemicals and biological activities of Citrus x microcarpa

Bunge revealed that the plant had an an antibiotic, antibacterial, antioxidant, and

antifeedant activities. These studies also revealed the presence of various compounds,

including flavonoids, alkaloids, polyphenols, and monoterpene hydrocarbons like

limonene, sabinene, citronellal, linalool, and hedycaryol. Flavonoid substances were also

found to have antibacterial properties (Mahardika & Roanisca, 2020). Flavonoids

(anthocyanins and flavones), a class of naturally occurring compounds derived from

plants, were known for their biological activity and role as secondary antioxidant defense

mechanisms, contributing to the color of the plants (Alam et al., 2022). Citrus flavonoids

were linked to various health benefits, including antioxidant, anti-cancer, anti-

inflammatory, anti-aging, and cardiovascular protective properties (Abid et al., 2022).

Examples of terpenoids included carvacrol, citronellal, geraniol, thymol, and menthol

(Astuti et. al, 2022). Phenolic compounds, exemplified by catechol (1, 2-

dihydroxybenzene), resorcinol (1, 3-dihydroxybenzene), and hydroquinone (1, 4

dihydroxybenzene), functioned as antioxidants, protecting cells from free radical damage

and potentially reducing the risk of cancer by hindering tumor growth (Mamari, 2021).

Flavonoids and phenolics were recognized as the two main bioactive compounds acting

as primary antioxidants or free radical scavengers (Singh et al., 2020).

Dishwashing Liquid

Detergents were widely used for cleaning in both households and commercial

settings. They were classified as hard surface cleaners, laundry detergents, and

dishwashing detergents based on their applications. Despite advancements in food

production technology, food safety concerns remained a significant issue due to the
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prevalence of bacteria, fungi, and other harmful microbes (Dai et al., 2019). Commercial

liquid dishwashing detergents were typically foaming mixtures containing around 20%

surfactants and 80% various additives, mainly water. They were primarily used for

washing glasses, cutlery, plates, kitchen utensils, and other dishes (Borkovic et al., 2021).

Cleaning products have altered the relationship between humans and the environment.

The increasing global production and market demand for these products heightened the

importance of assessing their impact. Cleaning or disinfecting cleaning tools could be a

method to contain bacterial contamination, prevent its further spread in the kitchen, and

decrease waste because the tools would last longer.

Commercial Dishwashing liquid

Dishwashing often involves encountering a lot of stains and grease. However, with

the help of commercial dishwashing liquid, users could easily remove these unwanted

residues. Researchers conducted emulsion stability tests using regularly used soils and a

model containing triolein and oleic acid at typical dosages (0.1 to 10%), water hardnesses

(2 and 15dH), and temperatures (25 to 45°C). Commercial dishwashing liquid was used

in the tests, containing 29.9 wt% agents with a 2.8:1 anionic/amphoteric ratio. The results

showed that emulsions were unstable and insensitive to temperature changes when only

triolein was used. However, the addition of oleic acid improved the stability. This

formulation ensured that the Joy brand dishwashing liquid wouldn't harm or dry users'

hands while using the product (Pinheiro Da Silva Pinto, 2022).

Antimicrobial Activity

As stated in the study, similar to the fruit peels, the calamansi leaves were

underutilized by society. Both calamansi peel and leaves could be used as antioxidants

and antimicrobials, due to their essential oil content. The essential oil in the fruit's peels
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was demonstrated to have an antimicrobial impact on bacteria like Staphylococcus

aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis. Based on

the measurement of the inhibitory diameter, its antimicrobial activity demonstrated a

robust inhibitory effect on the bacteria Staphylococcus aureus, Pseudomonas aeruginosa,

Escherichia coli, and Streptococcus mutans. Methicillin-resistant Staphylococcus aureus

was particularly susceptible, exhibiting a powerful inhibitory response (Dachriyanus et al.,

2021).

Concentration

The study employed the diffusion agar method, utilizing wells with extract

concentrations of 50% and 100% to test for antibacterial activity. As Dudek-Wicher &

Kowalska-Krochmal (2021) pointed out, increased exposure or concentration at the

infection site enhances the likelihood of therapeutic success.

Zone of Inhibition

The zone of inhibition test was most frequently used to measure the sensitivity or

resistance of bacteria to known or potential antimicrobial agents. After the zone of

inhibition of diffusion on the plate was calculated or the MIC of the antimicrobial agent was

determined, and the microbiologist confirmed that the quality control results indicated the

test system worked correctly, the results of the susceptibility tests needed to be

interpreted. Results for the majority of antimicrobial drugs were reported as either

susceptible, intermediate, or resistant and forwarded to the patient's chart (Tenover,

2019). An expansion of the zone of inhibition correlated with increasing AuNS

concentration treatments. The zone of inhibition demonstrated the effectiveness of the

AuNSs (Erdogan et al., 2023). Compounds with a significant inhibitory power had an
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inhibitory diameter of 10–20 mm, while those with a very strong inhibitory diameter had an

inhibitory diameter of more than 20 mm (Boukeskasse et al., 2019).

Salmonella spp.

Salmonella was the organism isolated from the intestine (Papa & Popa, 2021).

Salmonella enterica remained a major cause of foodborne illness in all parts of the world

with Typhimurium being one of the most common serotypes. The antigenic diversity of

lipopolysaccharides, flagellar proteins, and capsular polysaccharides provided the basis

for Salmonella serotyping. Most Salmonella strains were mobile and expressed the H

antigen, a peritrichous flagella. The expression of the fliC, fljBA, and hin genes controlled

the expression of the H antigen. The majority of Salmonella serotypes were biphasic,

meaning they could produce both phase 1 flagellin (FliC) and phase 2 flagellin (FljB) when

the H segment switched phases (Bai et al., 2019). The Enterobacteriaceae family contains

two species under Salmonella: S. enterica and S. bongori. The first had more than 2600

serotypes and six subspecies with Roman numbers that were differentiated according to

their somatic (O) and flagellar (H) features. S. enterica subspecies 1 was the most isolated

animal subspecies and was present in 99% of human isolates. Conversely, S. bongori

represented fewer than 1% of human isolates and was frequently detected in "cold-

blooded" species like fish, amphibians, and reptiles (Conte-Junoir et al., 2019). In middle-

and low-income nations, Salmonella enterica bacteria were the most frequent cause of

bloodstream infections. "Enteric or typhoid fever," a systemic febrile illness, was caused

by S. enterica serovar Typhi (S. Typhi), one of the human-restricted obligate pathogens

(Arshad et al., 2021). Humans and some larger primates were the sole reservoirs for these

bacteria. Symptoms such as diarrhea, fever, headaches, and abdominal pain could occur

within a week of incubation. Salmonellosis was recognized as the leading cause of

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gastroenteritis in humans, affecting both humans and animals and resulting in 93.8 million

cases and 155,000 fatalities annually (Conte-Junoir et al., 2019).

The authors discussed the C. microcarpa fruit, including its history, properties,

bioactive compounds, antimicrobial activity, concentration, and zones of inhibition. They

also addressed Salmonella spp. and commercial dishwashing practices. This citrus fruit

contained essential nutrients and compounds that were beneficial for health and had

pharmacological relevance. In addition to being widely sold commercially as juice, C.

microcarpa possesses anti-inflammatory, antioxidant, anthelmintic, antibacterial, and

antifungal properties, making it well-suited for cleaning kitchen utensils. Salmonella typhi,

a member of the Enterobacteriaceae family, could cause typhoid fever, a disease often

acquired through unsanitary kitchens. Modern consumers use dishwashing liquid to clean

dishes and combat pathogens. Available commercially, dishwashing liquid contains active

ingredients like surfactants, which produce foam and aid in the cleaning solution's

distribution to improve debris removal. Thickeners are also added to provide consistency,

making the detergent easier to handle and apply. Dishwashing liquid and other

components were influential in promoting food safety and lowering the risk of foodborne

diseases.

The researcher aimed to assess the effectiveness of C. microcarpa fruit peel

extract against Salmonella spp. Previous research established that calamansi peel

contains many phenolic compounds, including phenolic acids, tannins, and flavonoids.

These bioactive components within the C. microcarpa peel extract exhibit significant

antibacterial properties, demonstrating the potential to inhibit the growth of harmful

microorganisms effectively. The extracted materials were rigorously tested for

antibacterial sensitivity through a method specifically focused on evaluating the size of the

inhibitory zones formed around the extracts. Additionally, the study aimed to compare
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commercial dishwashing liquid to a formulated dishwashing liquid containing C.

microcarpa fruit extract, evaluating their effectiveness in eliminating Salmonella spp.

The researcher seeks answers to whether the antimicrobial activity of C. microcarpa

fruit peel is enough to protect against foodborne disease. Moreover, it aims to formulate

dishwashing liquid that is comparable to the standards of commercial dishwashing liquid

and health protocols from health agencies such as the Department of Health (DOH).

The purpose and objective of this study are to provide information for future

researchers and impart ideas for another possible alternative dishwashing liquid that is

organic based and can help impede harmful microorganisms.

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Chapter 3

METHODOLOGY

This chapter discussed the research design, research settings, research

instruments, data procedure and process flow, and data analysis that were relevant and

used during the examination.

Research Design

This study utilized an experimental design that involved various qualitative

research methods to gather information and provide a suitable interpretation of a specific

topic related to the antibacterial activity and effectiveness of dishwashing liquid made from

C. microcarpa fruit peel extract that inhibits Salmonella spp. The research study had two

primary components: modifying an independent variable and randomly assigning subjects

(Miller et al., 2020). The experimental design of this study allowed for precise variable

control and treatment comparison, both of which were necessary for a thorough

investigation of the antibacterial activity of C. microcarpa fruit peel extract-based

dishwashing solutions against Salmonella spp. This investigation was intended to assist

researchers in their work.

Research Setting

A study was conducted to determine the efficacy of dishwashing liquid made from

C. microcarpa fruit peel extract in treating Salmonella spp. The study was conducted at

Max Suniel Street, Carmen, 9000, inside PHINMA-Cagayan de Oro College, on the fifth

floor of the school's Main South Building, under the supervision of Mr. Joshua Richard

Payopanin Jr., Laboratory Instructor.

 23

Several laboratories at Central Mindanao University, University Town, Musuan,

Maramag, and Bukidnon were used to test the product. The plant used for the extract was

verified at the University Museum and underwent phytochemical testing at the Natural

Products Research and Development Center (NPRDC). The dishwashing liquid also

underwent antimicrobial testing at the Microbiology Laboratory of Veterinary Medicine.

The PHINMA-Cagayan de Oro College Laboratory was chosen for the formulation

of the extract and dishwashing liquid due to its availability of equipment, materials, and

apparatus. Meanwhile, Central Mindanao University was chosen for testing due to its

supply of apparatus, reagents, and technology necessary for the study. The researcher

aimed to obtain reliable and efficient results for the completion of the study.

Figure 2. PHINMA-Cagayan de Oro College CAHS Laboratory Room

 24

Map

source;https://www.google.com/maps/search/map+of+cmu+mindanao/@7.85882
55.1250502078.1700m/data=!3m1!1e3?entry:ttu

Figure 3. Central Mindanao University School Map

Research Instrument

An experiment was carried out using a dishwashing solution containing C.

microcarpa fruit peel extract to determine its impact on Salmonella spp. A single droplet

of liquid dishwashing solution impregnated with C. microcarpa fruit peel extract was added

to the cultured Salmonella spp. to assess its susceptibility. The strain used to examine the

efficiency of this mix was produced at Central Mindanao University's Microbiology

Laboratory.

Furthermore, the researcher performed the distillation process to extract a product

from the C. microcarpa fruit peel, and the end product was an essential oil.
 25

Figure 4: Distillation Process

Materials

● 1 unit weighing scale. It was used to weigh all of the ingredients included in the

dishwashing liquid composition.

● Two pcs. Plastic containers w/ wide mouth (1.5 liters capacity). It was used to

combine the various chemicals and materials used in the production of

dishwashing liquid. In contrast, one plastic container was used to mix the reagents

with dishwashing solutions, and the other container held the extract of C.

microcarpa fruit peel (calamansi).

● Stirring rod. It was used in stirring the reagents.

 26

● Plastic/glass bottle with cover (250 ml capacity/500 ml capacity/1 liter capacity/1

gallon capacity. It had been used as a storage bottle in the finished product of

dishwashing liquid derived from C. microcarpa (calamansi) extract.

 Thermometer. It was used to measure temperature. When the temperature

reaches 100 degrees Celsius, the process has ended.

● Distillation flask. It is primarily used for distillation, the process of separating a

mixture of C. microcarpa with different boiling points through evaporation, and

condensation.

● Condenser. It removes heat from the heated flask with C. microcarpa until it

condenses into a saturated liquid state.

 Reliability. It refers to the consistency of a measurement.

 Validity. It refers to accuracy.

Ethical Considerations

During the research, technical considerations were of utmost importance. It was

crucial to handle the extract and Salmonella spp. properly to ensure the safety of the

researchers and laboratory personnel. Therefore, certain procedures were followed.

The ethical considerations were connected to the research throughout the study.

 No animals or humans were harmed during the research experiment.

 All researchers who handled Salmonella spp. or the C. microcarpa fruit peel

extract-based dishwashing liquid were adequately trained in biosafety protocols.

This included using personal protective equipment (PPE) such as gloves, lab

coats, and goggles, to prevent accidental exposure.

 Proper waste disposal protocols were implemented for any materials contaminated

with Salmonella spp.

 27

 The privacy and security of all personal or sensitive information gathered during

the study were protected. The data storage, management, and sharing followed

applicable data protection requirements.

Data Gathering Procedure

Pre-Implementation

The researcher gathered all the letter to conduct the study at PHINMA Cagayan

de Oro College and permits to use the laboratory in formulating the dishwashing liquid

derived from C. microcarpa peel extract. It included a letter for the laboratory room used,

permission to borrow laboratory equipment, and a letter to the clinical laboratories

conducting the testing of the sample, In the formulation of the dishwashing liquid and

extract from C. microcarpa the followings materials are used; a unit weighing scale, plastic

container, stirring rod, glass bottle, with a cover, and other necessary instruments. The

bacteria used in the experiment was Salmonella spp. and was cultured and maintained

using nutrient agar. The effectiveness of the dishwashing liquid was measured using the

zone of inhibition at different concentrations.

Letter of Consent to Conduct the Study

Letter to the Laboratory for Extract Formulation

Borrowing Letter to Pharmacy Laboratory

Letter to the Laboratory for Antimicrobial Testing

Letter to the Laboratory for Phytochemical Testing

Figure 5: Pre-Implementation Flowchart

 28

During Implementation

The researcher formulated the dishwashing liquid derived from C. microcarpa fruit

peel extract at Max Suniel Street, Carmen, 9000, inside PHINMA Cagayan de Oro

College, located on the fifth floor of the Main South building. The researcher follows strict

adherence to the protocol and standard procedure imposed in the laboratory.

2 kilogram of Citrus microcarpa

Extraction of C. microcarpa by distillation process

Extract product mix with dishwashing liquid mixture

Testing of Dishwashing Liquid derived from C.

microcarpa fruit peel extract.

Figure 6. Flowchart of the Study from Sample Collection and Extract Formulation.

A. Extract formulation

1. The researcher gathered C. microcarpa fruit peel extract or calamansi plants at the

local market in Carmen, Cagayan De Oro City, Misamis Oriental.

2. The plant will be washed with clean tap water.

3. The slits had been cut in the lower ends of the fruits to remove seeds and juice.

4. Calamansi peel had been purified in this process, and the essential oil and distilled

water had been identified.

5. The first things taken and set up are a thermometer, a distillation flask, and a

condenser.

6. Remove the thermometer and fill the distillation flask with a boiling point chip,

approximately 30 grams of calamansi peel, and 200 ml of distilled water via a long-
 29

stem funnel. Begin boiling the calamansi peel. Once it starts boiling, reduce the

heat gradually.

7. Keep a tight eye on the temperature while distilling and gathering the distillate

when it reaches the condenser, which should be at 100 degrees Celsius. In this

range, the temperature ought to have been practically constant.

8. The temperature range across which you collected the calamansi essential oil

distillate should be noted in this step.

B. Dishwashing liquid formulation

1. In a mixing container, the distilled water was poured.

2. The S.A.A/Fortified Bubble Mixture was poured into the distilled water in a circular

hand motion. A small cut was made at the tip of the pouch, and the surfactant was

slowly poured out in one circular direction. It was allowed to sit for 20 minutes

before mixing.

3. It was stirred and mixed well.

4. The degreasing agent, scented chelating agent with antibacterial properties, and

pigment powder were added to the mixture. The same stirring process was

repeated carefully.

5. The C. microcarpa peel extract was added.

6. Once the mixture was well incorporated, the thickener was slowly poured. It wasn't

stirred quickly to ensure the thickener was dissolved.

7. Once the desired viscosity was achieved, the container was covered and left for

24 hours.
 30

C. Testing of the product

The researchers submitted the formulated dishwashing liquid derived from C.

microcarpa fruit peel extract at Central Mindanao University. There were two tests carried

out; a qualitative examination of C. microcarpa extract and the antibacterial activity of a

dishwashing liquid made from C. microcarpa extract inhibits Salmonella spp.

Dishwashing Liquid Derived from C. microcarpa peel extract against Salmonella

spp.

A. Sensitivity Testing B. Phytochemical Testing

Figure 7. Flowchart of the Study for Sample Testing

A. Sensitivity testing

The researcher will submit two dishwashing liquids: established dishwashing liquid

and dishwashing liquid made from C. microcarpa fruit peel extract. It will be submitted to

the Central Mindanao University. The laboratory will conduct sensitivity tests to determine

the extract's minimum inhibitory concentration (MIC), which kills the germs. Each sample

undergoes three trials.

 31

The researcher submitted the sample to the Microbiology laboratory at Central

Mindanao University and performed sensitivity testing.

They soaked filter paper discs in the sample/extract for 24 hours to reach their
maximum absorption.

The bacterial suspension was prepared by mixing a loopful colony of desired

bacteria with 5 mL of nutrient broth. The clarity of the bacterial suspension was
compared to a 0.5 McFarland Standard.

Using a sterile swab, the bacterial suspension was inoculated onto Mueller
Hinton (MH) Agar. The swab was dipped into the prepared bacterial suspension
and then streaked across the entire MH Agar surface.

The soaked filter paper disc from the sample/extract, antibiotic disc for positive
control, and filter paper disc soaked in distilled water for negative control were
carefully placed on the top surface of the inoculated MH agar.

The MH agar was then incubated for 18-24 hours at 37°C. The researcher
observed a zone of inhibition surrounding the filter paper disc and measured and
recorded the results.

Figure 8. Sensitivity Testing Process Flow

B. Phytochemical testing

The National Product Research and Development Center (NPDRC) at

Central Mindanao University handled the procedure. D.1 went on to discuss the

methods involved in identifying flavonoids and phenolic compounds using the

standard Shinoda Test (Panchal, 2019), whereas D.2 described the standard

Salkowski test, which was used to detect terpenoid compounds (Abdalla et al.,

2020).
 32

Citrus sp. extract fruit

Extract bioactive compounds at Central Mindanao University

Identification of Identification of Identification of

Flavonoids Terpenoids Phenolic compounds
compounds compounds

Figure 9. Phytochemical Testing Process Flow

B.1. Identification of the Flavonoid and Phenolic compound

1. 5mg of extract was added to the test tube, and then a tiny amount of magnesium

was mixed into this solution.

2. A few drops of concentrated Hydrochloric acid were added.

3. It revealed the presence of flavonoids by the colors pink, orange to red, magenta,

and red to crimson. Meanwhile, in the presence of phenolic chemicals, solutions

of greenish-black, brownish-green, black, and blue were formed.

B.2. Identification of Terpenoid Compound

1. 5 ml of extract was mixed with 2 ml of chloroform.

2. Carefully adding 3 millilitres of concentrated hydrogen peroxide created a layer.

3. A reddish-brown coloration of the interface was formed, showing positive results

for the presence of terpenoids.

 33

Post Implementation

This section assesses the efficacy of dishwashing liquid infused with C. microcarpa

fruit peel extract against Salmonella spp. To evaluate the dishwashing liquid's antibacterial

effectiveness under diverse circumstances, it was carried out several investigations.

Wherein, in the post-implementation of the study, the samples were stored in the

refrigerator for future purposes and the researchers measured the antimicrobial activity

based on the zone of inhibition measurements and provided a result and discussion.
 34

Chapter 4

FINDINGS, RESULTS, AND DISCUSSION

This chapter presented the findings, analysis, and interpretation of data gathered

whose main objective was to assess the effectiveness of dishwashing liquid derived from

C. microcarpa fruit peel extract in inhibiting and reducing the growth of Salmonella spp.

bacteria commonly found on kitchen surfaces and dishware.

Objective 1. To identify the bioactive compounds present in the C. microcarpa fruit peel
extract.
Table 1. Phytochemical Test of the Bioactive Compound of C. microcarpa Fruit Peel
Extract.

Bioactive compounds Sample Standard Reference Test Method

Flavonoids (-) Formation of Formation of Shinoda Test

yellowish solution red/magenta/pink-red
solution

Phenolic/Tannis (+) Formation of Formation of greenish- Shinoda Test

blackish solution black, brownish-green,
black, blue solution

Terpenoids (+) Formation of Formation of reddish- Salkowski Test

reddish-brown brown interphase
interphase

Legend:
(+) – Presence of the compounds
(-) – Absence of the compounds

Table 1 shows the qualitative identification of the bioactive compound of C.

microcarpa fruit peel extract was tested in the Center for Natural Products Research,

Development, and Extension at Central Mindanao University. The testing followed the

standard test methods, such as the Shinoda test for flavonoids, the phenolic/tannis test,

and the Salkowski test for terpenoids. In addition, it followed standard reference for the

interpretation of the result, wherein the flavonoid compound had the formation of a
 35

red/magenta/pink-red solution, the phenolic/tannis had the formation of a greenish-black,

brownish-green, black, and blue solution, and the terpenoids had the formation of a

reddish-brown interphase. Wherein the test yields a positive result in the bioactive

compound, such as phenolic or tannis, having a blackish solution and terpenoids having

a reddish-brown interphase. However, the C. microcarpa fruit peel extract yielded a

negative result in flavonoids having a yellowish solution.

In the study from Abdallah et al., (2023), it was stated that in recent years, phenolic

compounds and terpenoids have demonstrated significant antibacterial activity,

particularly through membrane disruption mechanisms, protein binding, interference with

intermediate metabolism, anti-quorum sensing, and anti-biofilm activity.

Phenolic compounds offer numerous health benefits. In terms of antibacterial activity,

hundreds of plants demonstrated antibacterial phenolic compounds extracted from

various plant parts, such as pomegranate peeled. The methanol, ethanol, and aqueous

extracts showed a high content of phenolic compounds and demonstrated remarkable

antibacterial activities against Staphylococcus aureus, Enterobacteraerogenes, Klebsiella

pneumoniae, and Salmonella typhi. Terpenoids were frequently utilized in the production

of tastes, perfumes, insecticides, pharmaceuticals, and industrial chemicals. According to

numerous studies, many terpenoid molecules have significant antibacterial action. Twelve

pure terpenoid compounds extracted from the wood and bark of an aromatic cypress tree

(Pilgerodendronuviferum) demonstrated varying degrees of remarkable antibacterial

activity against twelve different microorganisms, including Staphylococcus aureus,

Bacillus subtilis, Streptococcus pyogenes, Escherichia coli, Pseudomonas aureginosa,

and Fusariumgraminea (Abdallah, E. & Sadeek, A., 2019). On the contrary, when polar

groups, such as hydroxyl and glycosyl, were inserted into the structures of flavonoids, their

inhibitory effects against gram-positive bacteria decreased. This could be viewed as

 36

flavonoids' excessive hydrophilicity preventing their entry into phospholipid bilayers and

contact with the cell membrane's hydrophobic area. (Cao et al., 2021).

Objective 2 To measure the antimicrobial activity of dishwashing liquid derived from C.

microcarpa fruit peel extract at different concentrations (50%, 70%, and 100%) against
Salmonella spp. as measured through the zone of inhibition.
Table 2. Zone of Inhibition at 50%, 70%, and 100% of the Dishwashing liquid derived
from C. microcarpa Fruit Peel Extract.

Dishwashing Liquid with C. Zone of Inhibition (mm2) Average Interpretation

microcarpa peel extract
Number of Trials

1 2 3 4 5 6

50% 8.0 8.1 7.6 8.3 8.0 7.4 7.9 Sensitive

70% 8.4 8.3 8.5 8.1 8.4 8.7 8.4 Sensitive

100% 7.2 7.5 7.7 8.3 8.2 8.2 7.9 Sensitive

Legend:
Sensitive – A zone of inhibition appears on the agar plate.
Resistant – No zone of evidence.

Table 2 shows the zone of Inhibition at 50%, 70%, and 100% of the Dishwashing

liquid derived from C. microcarpa fruit peel extract. The sensitivity test result showed in

the figure above that dishwashing liquid had an inhibitory effect on Salmonella spp.

growth. A greater zone of inhibition was shown in the 70% concentration of dishwashing

liquid derived from C. microcarpa fruit peel extract, as indicated by the 1 (Trial 1) value of

8.4 mm2, 2 (Trial 2) value of 8.3 mm2, 3 (Trial 3) value of 8.5 mm2, 4 (Trial 4) value of 8.1

mm2, 5 (Trial 5) value of 8.4 mm2, and 6 (Trial 6) having a value of 8.7 mm2, which had an

average zone of inhibition of 8.4 mm2 in all trials, stipulating higher sensitivity in inhibiting

the growth of the Salmonella spp. Additionally, 50% concentration and 100% showed
 37

sensitivity activity of the bacteria with 7.9 mm2 in both concentrations, which was lower

than the 75% concentration.

Based on the concept presented by Giuliano et al., 2019, according to CLSI

criteria, the diameter of the area with no growth was measured to determine susceptibility.

Larger zones implied less bacterial growth and more antibiotic susceptibility, whereas

smaller zones suggested more bacterial growth and lower antibiotic susceptibility. There

was no zone of inhibition if the antibiotic did not limit growth; thus, the bacteria were

resistant. Based on the results presented, Salmonella spp. was sensitive to dishwashing

liquid derived from C. microcarpa fruit peel extract, having the highest degree of

antimicrobial activity with 70%, followed by 100% and 50%. Calamansi lime essential oil

was shown to have antibacterial activity against tested bacterial pathogens such as E.

coli, Salmonella spp., B. subtilis, and Streptococcus spp., each with a different inhibitory

zone (Alias et al., 2020). The mechanisms of action (MOAs) of EO antimicrobial activities

were studied from the standpoint of interactions between EOs and target microorganisms.

Disrupting the outer membrane; causing cell lysis or the release of lipopolysaccharides;

changing the fatty acid composition of the membrane; dissolving, aligning, or forming

channels in the phospholipid bilayer; interfering with or inhibiting glucose uptake; and

inhibiting enzyme activity were examples of these principles that inhibited the growth of

the bacteria (Cho et al., 2020).

 38

Objective 3. To compare the effectiveness of dishwashing liquid derived from C.

microcarpa fruit peel extract in different concentrations and commercial dishwashing liquid
against Salmonella spp.
Table 3. Comparison between Dishwashing liquid derived from C. microcarpa Fruit Peel
Extract to Commercial dishwashing Liquid at Different Concentrations.

Testing Sample Average Zone of Inhibition (mm2)

50% 70% 100%

Dishwashing Liquid with C. 7.9 8.4 7.9

microcarpa fruit peel extract

Commercial Dishwashing 6.3

liquid

Ciprofloxacin (+ control) 25

Legend:
Sensitive – A zone of inhibition appears on the agar plate.
Resistant – No zone of evidence.

Table 3 shows the study comparison of the dishwashing liquid derived from C.

microcarpa fruit peel extract to commercial dishwashing liquid in terms of the zone of

inhibition and concentration. The sensitivity testing results showed that dishwashing liquid

derived from C. microcarpa peel fruit extract was effective, having a zone of inhibition in

millimeters with 7.9 mm2 in 50% concentration and 100% concentration and 8.4 mm2 in

70% concentration, interpreted as sensitive and capable of inhibiting the growth of the

bacteria, Salmonella spp. On the other hand, the sensitivity testing of commercial

dishwashing liquid showed lower antimicrobial activity compared to extract-infused

dishwashing liquid, with a zone of inhibition of 6.25 mm2, which is lower than 7 mm2.

Additionally, the outcome of the positive control was displayed. Ciprofloxacin (the positive
 39

control) had a thickness of 25 mm2. Based on the findings, ciprofloxacin inhibits bacterial

growth strongly, as shown by the comparatively broad zone of inhibition of 25 mm2.

The dishwashing liquid infused with C. microcarpa peel extract demonstrated

higher effectiveness, as indicated by larger zones of inhibition at various concentrations

(7.9 mm2, 8.4 mm2) against Salmonella spp. compared to the commercial dishwashing

liquid (6.25 mm2). These findings suggested that the extract-infused dishwashing liquid

was more sensitive and capable of inhibiting bacterial growth, highlighting its potential as

an alternative cleaning solution with strong antimicrobial properties. According to research

by Alam et al., (2022), citrus fruits could be used as a natural antibiotic in place of

manufactured ones. The physicochemical features of the liquid derived from the three

citrus fruits produced locally indicated that it was of good quality. The results suggested

that citrus fruits could be used as a valuable source of components that promoted health.

Lemons are an effective natural element for cleaning and disinfecting household surfaces,

especially kitchenware, due to their multifunctional capabilities, which include

antibacterial, antifungal, and antioxidant qualities (Sabir et al., 2021). Because they

offered cleaning performance comparable to conventional products but were safer and

healthier, eco-friendly dishwashing liquids could make a substantial contribution to a more

sustainable and healthful future (Gómez et al. 2019).

 40

Objective 4. To determine the bioactive compounds present in dishwashing liquid derived

from C. microcarpa fruit peel extract at different concentrations, significantly influence both
the antimicrobial activity and effectiveness of C. microcarpa fruit peel extract in
comparison to commercial dishwashing liquid against Salmonella spp.

Table 4. Interpretation of Sensitivity Testing.

Type of Dishwashing Liquid Average Zone Interpretation

of Inhibition
2
(mm )

Dishwashing liquid derived from C. microcarpa (50%) 7.9 Sensitive

Dishwashing liquid derived from C. microcarpa (70%) 8.4 Sensitive

Dishwashing liquid derived from C. microcarpa (100%) 7.9 Sensitive

Commercial dishwashing liquid 6.3 Sensitive

Legend:
Sensitive – A zone of inhibition appears on the agar plate.
Resistant – No zone of evidence.

Table 4 shows the interpretation of Sensitivity Testing of two types of dishwashing

liquid; the dishwashing liquid derived from C. microcarpa fruit peel extract and a

commercial dishwashing liquid. The results revealed that among all dishwashing liquids,

the dishwashing liquid derived from C. microcarpa fruit peel had the highest zone of

inhibition of 8.4 mm2, indicating its potency in eliminating bacteria. It was followed by 50%

and 100%, both having a zone of inhibition of 7.9 mm2. The commercial dishwashing liquid

had a smaller zone of inhibition of 6.3 mm2, indicating less potency and synergistic activity

against bacteria, including Salmonella spp. According to Microhem Laboratory (n.d),

bacterial strains were susceptible to antimicrobial agents if a zone of inhibition appeared

 41

on the agar plate, while no zone was shown if they were resistant to the antimicrobial

agent.

The outcomes of the two types of dishwashing liquid that underwent sensitivity

testing, one of which was the dishwashing liquid derived from C. microcarpa fruit peel

extract and the commercial dishwashing liquid, offered important insights regarding their

antibacterial activity. When compared to its commercial counterpart, the dishwashing

liquid derived from C. microcarpa fruit peel extract showed the biggest zone of inhibition,

measuring 8.4 mm2. This suggested that it was more potent in removing germs. This result

was consistent with other studies that had demonstrated the potential antibacterial

qualities of natural extracts, like those made from citrus fruits (Alam et al., 2022).

The observed trend of increasing zone of inhibition with higher concentrations of

the C. microcarpa fruit peel extract-infused dishwashing liquid suggested a dose-

dependent effect on antimicrobial activity. The presence of these bioactive compounds in

the extract may have contributed to its ability to inhibit the growth of Salmonella spp. by

disrupting essential cellular processes or structures. Additionally, the significantly higher

zone of inhibition exhibited by the C. microcarpa fruit peel extract-infused dishwashing

liquid compared to the commercial counterpart suggested that the bioactive compounds

present in the extract played a crucial role in enhancing its antimicrobial effectiveness. It

offers an accurate basis for making a substance that can stop bacteria from growing.

Citrus fruits can therefore be utilized as a natural antibiotic in place of manufactured ones

(Alam et al., 2022).

Ultimately, the bioactive compounds present in dishwashing liquid derived from C.

microcarpa fruit peel extract significantly influenced its antimicrobial activity and

effectiveness against Salmonella spp. The concentration-dependent increase in

antimicrobial efficacy, along with the superior performance compared to the commercial
 42

dishwashing liquid, highlighted the potential of natural extracts as effective antimicrobial

agents for household disinfection and bacterial control (Ahmad et al., 2023).
 43

Chapter 5

SUMMARY, CONCLUSION, RECOMMENDATION

This chapter presented the summary, conclusions, and recommendations

drawn from the experiment conducted and the analysis of the results.

Summary

The primary objective of the research was to assess the effectiveness of a

dishwashing liquid formulated with C. microcarpa fruit peel extract in inhibiting and

reducing the growth of Salmonella spp. Bacteria are commonly found on kitchen

surfaces and dishware. The study included specific objectives such as validating and

producing an extract from Citrus spp., formulating a dishwashing liquid incorporating

C. microcarpa fruit peel an extract, and determining the antimicrobial activity of the

extract versus a commercial dishwashing liquid. The effectiveness of the formulated

dishwashing liquid was measured using the zone of inhibition of Salmonella spp.

Growth between three different concentrations. The research also aimed to compare

the effectiveness of the C. microcarpa extract-infused dishwashing liquid derived from

a commercial brand, regarding the diameter of the zone of inhibition and the

concentration of the dishwashing liquid.

Moreover, the study's theoretical framework was based on the Germ theory and

the principle of antibiotic susceptibility testing, which helped determine the efficacy of

the dishwashing liquid against Salmonella spp. using the oil extracted from the fruit

peel of C. microcarpa. Since the independent variable in the study was the bioactive

compound in dishwashing liquid derived from C. microcarpa fruit peel extract

(Flavonoids, Terpenoids, and Phenolic/Tannis), concentrations of C. microcarpa fruit

 44

peel extract (50%, 70%, and 100%) and the type of dishwashing liquid. The dependent

variables were the antimicrobial activity and effectiveness of dishwashing liquid

derived from C. microcarpa fruit peel extract against Salmonella spp. and measured

by the zone of inhibition with ciprofloxacin as control. The data analysis involved

submitting the extract to the Natural Product Research and Development Center

(NPRDC) for bioactive compound identification and conducting antimicrobial testing at

the Microbiology Department at Central Mindanao University. The research instrument

included a dishwashing solution containing C. microcarpa peel extract, and the

procedure involved formulating the dishwashing liquid, using a distillation process to

obtain the essential oil from the fruit peel, and testing the cultured Salmonella spp. for

susceptibility.

Results of the tests indicated that the dishwashing liquid derived from Citrus

microcarpa fruit peel extract effectively inhibited the growth of Salmonella spp. The

extract-infused dishwashing liquid derived from C. microcarpa peel extract showed a

zone of inhibition of 7.9 mm2 at both 50% and 100% concentrations, and 8.4 mm2 at

70% concentration, which were interpreted as sensitive and capable of inhibiting

bacterial growth. In comparison, the commercial dishwashing liquid was found to have

less antimicrobial activity with a zone of inhibition of 6.3 mm2. The positive control,

Ciproflaxin, showed a robust inhibitory effect with a zone of inhibition of 25 mm 2.

Conclusion

The study's findings led to the following conclusions being made:

1. The dishwashing liquid formulated with Citrus microcarpa fruit peel extract was

effective in inhibiting and reducing the growth of Salmonella spp.

 45

2. The extract from Citrus microcarpa fruit peel exhibited antimicrobial properties,

which, when incorporated into the dishwashing liquid, effectively killed or inhibited

the growth of Salmonella spp. commonly found on kitchen surfaces and dishware.

3. The bioactive compounds of the plant, such as Phenolics and Terpenoids,

contributed to the antimicrobial activity of the C. microcarpa in inhibiting the growth

of the bacteria.

4. Using dishwashing liquid derived from C. microcarpa extract could help combat

foodborne diseases acquired from utensils.

5. The community could save money by opting for organically produced that showed

higher antibacterial activity than commercial dishwashing liquid.

Recommendation

In line with the findings of the study, the following recommendations are made:

1. The Philippine Health Agency should consider mandating standard regulations for

including ingredients with antibacterial properties, such as terpenoids and

phenolics, in all dishwashing liquids. This would ensure that these products

effectively inhibit bacterial growth on kitchen utensils.

2. The Department of Agriculture should focus on enhancing crop production and

utilization of plants like Citrus microcarpa, which have health benefits and can

support local farmers.

3. Community members should prioritize using dishwashing liquids that are safe and

effective in eliminating microorganisms, especially Salmonella spp., which are

known to cause foodborne diseases.

 46

4. It is recommended that the community opts for dishwashing liquid formulated with

Citrus microcarpa extract to guarantee that kitchen utensils are thoroughly cleaned

and protected from pathogens.

5. Additional plant parts such as leaves, roots, and stems could be extracted and

tested as supplementary ingredients for dishwashing liquid formulations to assess

their ability to inhibit bacterial growth.

6. Future research endeavors could focus on conducting further antimicrobial studies

such as including citrus juice as an ingredient in making dishwashing liquid and its

effect on other strains of bacteria, particularly gram-positive bacteria. This is due

to the observed antibacterial activity of the extract, suggesting its potential for

broader applications in hygiene and sanitation.

 47

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Abdalla, A., Makhawi, A., & Mustafa, M. (2020). Phytochemical screening and
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Abdallah, E., & Sadeek, A. (2019). Phytochemical compound as antibacterial agents: A

mini review. Global Journal of Pharmacy & Pharmaceutical Sciences, 7(4).

Abdallah, E., Alhatlani, B., Martins, C., & Menezes, R. (2023). Back to nature: Medicinal
plants as promising sources for antibacterial drugs in the post-antibiotic era.
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Afriyandi, D., Husni, E., & Ismed, F. (2020). Standardization study of simplicial and extract
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Ahmad, M., Ahmed, M., Asari, A., Durani, A., Muddassar, M., & Saleem, M. (2023).
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 51

APPENDICES

APPENDIX A

Letter of Consent for Phytochemical Testing

 52

APPENDIX B

Letter of Consent for Antimicrobial Testing

 53

APPENDIX C

Letter of Consent to Laboratory for Extract Formulation

 54

APPENDIX D

Letter of Consent of Borrowing Pharmacy Laboratory Equipment

 55

Appendix E

Laboratory Procedure

I. Objectives: At the end of the laboratory activity, the researcher shall able to:

A. Make an essential oil from the extract of C. microcarpa fruit peel.

B. Make a dishwashing liquid mixture.

C. Have a product of dishwashing liquid derived from C. microcarpa fruit peel extract.

II. Materials

1. Laboratory apparatus

- Distillation Flask

- Condenser

- Erlenmeyer Flask 2 pieces

- Beaker

- Thermometer

- Bucket 2 pieces

- Stirring rod

- Mineral bottle 2 pieces

- Container 3 pieces

2. Ingredients

- Calamansi 2 kilogram

- Surfactant

- Thickener

- Foam booster

- Degreasing agent

- Scented chelating agent

- Pigment color
 56

- Distilled water 10Liters

III. Procedure

1. Extract formulation

- Gathered C. microcarpa fruit peel extract or calamansi plants.

- Washed the fruit with clean tap water.

- Cut the slits will be in the lower ends of the fruits to remove seeds and juice.

- A thermometer, distillation flask, and a condenser should be first be taken and set-

up.

- Take out the thermometer and put a boiling point chip and around 200 ml of

calamansi leaves in the distillation flask using long-stem funnel.

- Start boiling the calamansi leaves. Once it starts boiling turn down the heat so it

does so gradually.

- Keep a tight eye on the temperature while distilling and gather the distillate when

it reaches the condenser and is between 100 degrees Celsius. In this range, the

temperature ought to be practically constant.

- Immediately switching collecting flask when the temperature exceeds 80 degrees

Celsius and continue distilling until the temperature is between 98 and 100 degrees

Celsius.

- The temperature range across which you collected the calamansi essential oil

distillate should be noted in this step.

2. Dishwashing liquid formulation

- In a mixing container, pour the distilled water.

- Pour the S.A.A/Fortified Bubble Mixture into the distilled water in a circular hand

motion. Make a small cut at the tip of the pouch and slowly pour out the surfactant

in one circular direction. Let it sit for 20 minutes before mixing.

- Stir and mix it well.

 57

- Add the degreasing agent, scented chelating agent with antibacterial and pigment

powder into the mixture. Repeat the same stirring carefully.

- Add the C. microcarpa peel extract.

- Once mixture is well incorporated, slowly pour the thickener. Do not stir it in a fast

motion. Make sure thickener is dissolved. \

- Once desired viscosity is reached, cover the container, and leave it for 24 hours.

3. Identification of the Flavonoid compound\

1. 5mg extract was added in the test tube then small amount of magnesium was mixed

in this solution.

2. Add the few drops of concentrated Hydrocloric acid.

3. It will indicate the pink colour, orange to red, magenta, and red to crimson with

presence of flavonoids. While, formation of greenish-black, brownish-green, black,

blue solution with presence of phenolic compound.

4. Identification of the terpenoid compound

1. 5 ml of extract mixed with 2 ml of chloroform.

2. 3 ml concentrated H2SO4 was carefully added to form a layer.

3. A reddish-brown coloration of the interface was formed to show positive results for

the presence of terpenoids.

5. Sensitivity Testing

1. Soak filter paper disc into the sample/extract for 24hours, to reach its maximum

absorption.

2. Prepare bacterial suspension by mixing a loopful colony of desired bacteria to a

5mL nutrient broth. (Compare the clarity of your bacterial suspension to a 0,5

McFarland Standard).
 58

3. Using a sterile swab, inoculate your bacterial suspension to a Mueller Hinton (MH)

Agar. Dip the swab to the prepared bacterial suspension then streak it to the entire

MH agar surface.

4. Carefully place your soaked filter paper disc from your sample/extract, antibiotic

disc for positive control, and filter paper disc soaked in distilled water for negative

control at the top surface of inoculated MH Agar. (Incubate MH Agar for 18-24

hours at 37 degrees Celsius)

5. Observe for a zone of inhibition surrounding your filter paper disc. Measure and

record the result

 59

APPENDIX F

LABORATORY DATA

DATE: November 16, 2023

BATCH 1 BATCH 2

WATER: 200 mL WATER: 200 mL

PEEL: 35 g PEEL: 35 g

TIME START: 10:04 Am TIME START: 11:20 AM

TIME END: 10:55 AM TIME END: 2:15 PM

mL EXTRACTED: 20 mL mL EXTRACTED: 20 mL

LITMUS PAPER: Turn pale pink LITMUS PAPER: Turn pink in

second

BATCH 3 BATCH 4

WATER: 200 mL WATER: 200 mL

PEEL: 35 g PEEL: 35 g

TIME START: 2:05 Pm TIME START: 6:30 Pm

TIME END: 5:30 PM TIME END: 7:50 Pm

mL EXTRACTED: 30 mL mL EXTRACTED: 10 mL

LITMUS PAPER: Turn pink in second LITMUS PAPER: Turn pink

in second

TOTAL WATER USED: 800 mL

TOTAL PEEL USED: 140 grams

TOTAL EXTRACTED : 60 mL (discarded the Batch 1)

SAMPLE FOR PHYTOCHEMICAL TESTING

 60

DATE: DECEMBER 18, 2023

BATCH 1

SET-UP 1 SET-UP 2

WATER: 200 mL WATER: 200 mL

PEEL: 50 g PEEL: 50 g

TIME START: 07:10 Am TIME START: 7:10 AM

TIME END: 10:20 AM TIME END: 10:20 PM

mL EXTRACTED: 55 mL mL EXTRACTED: 55 mL

LITMUS PAPER: Turn pink in second LITMUS PAPER: Turn pink in

second

BATCH 2

SET-UP 1 SET-UP 2

WATER: 200 mL WATER: 200 mL

PEEL: 50 g PEEL: 50 g

TIME START: 10:25 Pm TIME START: 10:25 Pm

TIME END: 1:35 PM TIME END: 1:50 Pm

mL EXTRACTED: 60 mL mL EXTRACTED: 60 mL

LITMUS PAPER: Turn pink in second LITMUS PAPER: Turn pink

in second
 61

BATCH 3

SET-UP 1 SET-UP 2

WATER: 200 mL WATER: 200 mL

PEEL: 50 g PEEL: 50 g

TIME START: 1:45 Pm TIME START: 2:00 AM

TIME END: 4:30 Pm TIME END: 5:10 PM

mL EXTRACTED: 60 mL mL EXTRACTED: 50 mL

LITMUS PAPER: Turn pink in second LITMUS PAPER: Turn pink in

second

BATCH 4

SET-UP 1 SET-UP 2

WATER: 200 mL WATER: 200 mL

PEEL: 50 g PEEL: 50 g

TIME START: 4:35 Pm TIME START: 5:25 Pm

TIME END: 7:30 PM TIME END: 8:30 Pm

mL EXTRACTED: 55 mL mL EXTRACTED: 55 mL

LITMUS PAPER: Turn pink in second LITMUS PAPER: Turn pink

in second

CONCENTRATIONS

200 𝑚𝐿 (𝑒𝑥𝑡𝑟𝑎𝑐𝑡) 140 𝑚𝐿 (𝑒𝑥𝑡𝑟𝑎𝑐𝑡)

v/v % = 200 𝑚𝐿 (𝑑𝑖𝑠ℎ𝑤𝑎𝑠ℎ𝑖𝑛𝑔 𝑙𝑖𝑞𝑢𝑖𝑑) 𝑥 100 v/v % = 200 𝑚𝐿 (𝑑𝑖𝑠ℎ𝑤𝑎𝑠ℎ𝑖𝑛𝑔 𝑙𝑖𝑞𝑢𝑖𝑑) 𝑥 100

= 100 % = 70%

100 𝑚𝐿 (𝑒𝑥𝑡𝑟𝑎𝑐𝑡)
v/v % = 𝑥 100 = 50
200 𝑚𝐿 (𝑑𝑖𝑠ℎ𝑤𝑎𝑠ℎ𝑖𝑛𝑔 𝑙𝑖𝑞𝑢𝑖𝑑)

SAMPLES FOR SENSITIVITY TESTING

 62

Appendix G

BUDGET PROPOSAL

MATERIALS COST

Microcarpa fruit ( 4 kg) 240

Teflon (5 rolls/ 1/2 meters) 100

Flying tiger (4 bottles) 180

Styrofoam (1/8) 55

Level Hose (9 meters) 90

Container (3 bottles) 54

Commercial Dishwashing Liquid 81.85

Dishwashing set 110

Distilled water 5L 95

Test for C. microcarpa validation 180

Test for Extract-infused Dishwashing liquid to Salmonella 900

spp. (50%, 70%, 100%)

Test for Commercial Dishwashing liquid to Salmonella spp. 300

Bioactive testing for C. microcarpa extract (Flavonoid, 1,400

Terpenoid, & Phenolic)

Transportation (3 ways good for 2 persons) 2,020

TOTAL COST 5,805.85

Prepared by:

Aimei Pearl C. Alboradora

Research Leader
 63

APPENDIX H

Salmonella spp.. DATA

 64

APPENDIX I

PLANT VERIFICATION AND VALIDATION

 65

APPENDIX J

EXTRACT VALIDATION
66
 67

APPENDIX K

SENSITIVITY TESTING LABORATORY RESULT

68
69
70
 71

APPENDIX L

Phytochemical Testing Result

 72

APPENDIX M

DOCUMENTATION

EXTRACT FORMULATION SET-UP

73
 74

ON-GOING EXTRACTION
 75

PRODUCT

100 %

70 %

50 %
76
 77

100% DIishwashing Liquid 70% DIishwashing Liquid Derived

Derived from C. microcarpa Fruit from C. microcarpa Fruit Peel
Peel Extract Extract

50% DIishwashing Liquid Derived Commercial Dishwashing Liquid

from C. microcarpa Fruit Peel
Extract
 78

APPENDIX N

CURRICULUM VITAE

Name: Elmie Jane E. Abang

Address: Zone 7, Tagbao, Cogon, El Salvador City, Misamis Oriental
Email Address: eleg.abang.coc@phinmaed.com
Name of Father: Elmer L. Abang
Name of Mother: Elvie E. Abang
School Graduated and Honors
Elementary
Cogon Elementary School
 4th Honor
Junior High School
Cogon National High School
 With Honor
Senior High School
PHINMA-Cagayan de Oro College
College
PHINMA Cagayan de Oro College
 79

Name: Kate Hyacinth Abriam

Address: San Isidro Labrador, Agora, Lapasan, Cagayan de Oro City,
Misamis Oriental, 9000
Email Address: kate.abriam.coc@phinmaed.com
Name of Father: Alvin M. Valdez
Name of Mother: Marinel G. Abriam
School Graduated and Honors
Elementary
Kalilangan Elementary School
Junior High School
Misamis Oriental General Comprehensive High School
Senior High School
PHINMA-Cagayan de Oro College
College
PHINMA-Cagayan de Oro College
 80

Name: Aimei Pearl C. Alboradora

Address: Purok-3F, Upper Magsaysay, Kuya, Maramag, Bukidnon
Email Address: aica.alboradora.coc@phinmaed.com
Name of Father: Marvin B. Alboradora
Name of Mother: Analyn C. Alboradora
School Graduated and Honors
Elementary
Magsaysay Elementary School
 Valedictorian

Junior High School

Pangantucan National High School
 With High Honor
Senior High School
Central Mindanao University
 With Honor
College
PHINMA Cagayan de Oro College
 81

Name: Kaye Angelie N. Aljas

Address: Poblacion Kalawit, Zamboanga Del Norte
Email Address: kana.aljas.coc@phinmaed.com
Name of Father: Jose J. Aljas
Name of Mother: Vivien N. Aljas
School Graduated and Honors
Elementary
Kalawit Central School
 Salutatorian
Junior High School
St. Vincent’s College Incorporated
Senior High School
St. Vincent’s College Incorporated
 With High Honor
College
PHINMA Cagayan de Oro College
 82

Name: Kean Harvey E. Arellano

Address: P-1b, South Poblacion, Maramag, Bukidnon
Email Address: Kees.arellano.coc@phinmaed.com
Name of Father: Rodelo B. Arellano
Name of Mother: Arcelie E. Arellano
School Graduated and Honors
Elementary
San Vicente Elementary School
 First Honorable Mention
Junior High School
Father Urios High School Properidad
Senior High School
Bukidnon National High School of Home and Industries
College
PHINMA Cagayan de Oro College
 83

Name: Louise Brylle O. Balatayo

Address: GSIS Housing Zone 7, Calaanan, Canitoan, Cagayan de Oro City
Email Address: loom.brylle10@gmail.com
Name of Father: Marloue C. Balatayo
Name of Mother: Florinda O. Balatayo
School Graduated and Honors
Elementary
West City Central School
 With Honor
Junior High School
Misamis Oriental General Comprehensive High School
 With Honor
Senior High School
Misamis Oriental General Comprehensive High School
 With Honor
College
PHINMA Cagayan de Oro College
 84

Name: Joshua O. Balbutin

Address: Barangay 24, Gingoog City, Misamis Oriental
Email Address: jooc.balbutin.coc@phinmaed.com
Name of Father: Benjamin T. Balbutin
Name of Mother: Maria S. Oclaret
School Graduated and Honors
Elementary
Visayan Village Central Elementary School
 First Honorable Mention
Junior High School
Gingoog City Comprehensive National High School
 With Honor
Senior High School
Gingoog City Comprehensive National High School
 With High Honor
College
PHINMA Cagayan de Oro College
 85

Name: Shawney M. Barrita

Address: Zone 3, Baikingon, Cagayan de Oro City
Email Address: shma.barrita.coc@phinmaed.com
Name of Father: Colbin A. Barrita
Name of Mother: Rechel M. Barrita
School Graduated and Honors
Elementary
Baikingon Elementary School
 Top 10 Honor List
Junior High School
Iponan National High School
Senior High School
PHINMA Cagayan de Oro College
College
PHINMA Cagayan de Oro College
 86

Name: Rosh Hashana Louisse S. Matbagan

Address: Purok 8, Central Poblacion, Kalilangan, Bukidnon
Email Address: rosa.matbagan.coc@phinmaed.com
Name of Father: Roldan C. Matbagan
Name of Mother: Jackie Lou S. Matbagan
School Graduated and Honors
Elementary
Kalilangan Central Elementary School
 With Honor
Junior High School
Kalilangan National High School
 With Honor
Senior High School
San Agustin Institute of Technology
 With High Honor
College
PHINMA Cagayan de Oro College
 87

Name: Louilla Mae M. Naive

Address: Zone 2 Cugman, Cagayan de Oro City
Email Address: loma.naive.coc@phinmaed.com
Name of Father: Wilson L. Naive
Name of Mother: Lourdes M. Naive
School Graduated and Honors
Elementary
City Central School
 With Honor
Junior High School
Pilgrim Christian College
 With High Honor
Senior High School
Lourdes College
College
PHINMA Cagayan de Oro College