Using Oregano (Origanum Vulgare) Extract to Inhibit the Proliferation of Mango Molds

Cidro, Princess Hannah M. † Fernandez, Abegail Anne Z. † Llamado, Jino M.

Sta. Elena High School
W.C. Paz St., Sta. Elena, Marikina City

Abstract

Mango (Mangifera Indica) is a fruit that is known for its distinctive flavor, and several

health benefits. However, this fruit gets deteriorated easily since it can adapt different

microorganisms - especially molds - in the rapid changing of temperature, and post-harvest

activities. The purpose of this study was to find a product that can inhibit the growth of molds

that causes deterioration and spoilage in mangoes. Oregano (Origanum Vulgare) - a flowering

plant in the mint family (Lamiaceae) - is proven to have antimicrobial and antibacterial

properties. By using various methods, abundant phenols like thymol and carvacrol can be

obtained. Extracted mango molds were placed in a potato dextrose agar, and incubated for 5

days. Sterile disc-shaped filter papers were soaked in different concentrations of oregano, 30%,

60%, and 90%. These disc-shaped filter paper were put in the extracted mango molds, and zone

of inhibition was measured after 2 days. The mean of molds without the oregano extract was 0

mm while the mean of molds with oregano extract showed an alteration of 10.75 mm by its zone

of inhibition. Then the different concentration of oregano extract to the zone of inhibition of

molds was 11.33 mm in 30% concentration of Oregano extract, 13.67 mm in 60% concentration

of oregano extract, and in 90 % concentration of Oregano extract had a greater zone of inhibition

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of 18 mm. This showed that Oregano extract was capable of inhibiting the proliferation of molds

and increasing the concentration of oregano extract expands the zone of inhibition in molds.

Thus, the results showed that Oregano extract has a natural component suitable for use as an

antimicrobial agent and can lessen the usage of chemical food preservatives.

Keywords: Mango (Mangifera Indica), Oregano (Origanum Vulgare), incubation, extraction,

zone of inhibition, antimicrobial properties

Introduction

Mangoes (Mangifera Indica) are known for its unique flavor, fragrance, taste, and heath

promoting qualities. It is often labelled as “king of the fruits”, and “super fruits”. Mango is a tree

cultivated in many tropical regions such as India, Pakistan, Bangladesh, and the Philippines.

Now farming of Mango trees has been extended in many continents, not only because of the taste

fruit that consumers enjoy but also because of its numerous health benefits.

Nowadays, Mango (Mangifera Indica) gets spoiled easily – in which it host different

molds because of the changing temperature and post-harvest activity i.e. storage and handling

techniques (Al Najada, et al., 2014), to prevent this, a product is needed to inhibit the growth of

molds on mango (Mangifera Indica). Oregano (Origanum Vulgare) is an herb in which its

extract has demonstrated antimicrobial actions to different food-borne pathogens (Singletary,

2010).

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 This study assesses the oregano extract as an inhibitor for the growth of molds that causes

deterioration and spoilage of Mango fruits. Specifically, this evaluates cultivation and inhibition

of molds - Aspergillus niger, Aspergillus flavus, and Penicillium Spp.

This study can help to lessen the usage of chemical food preservatives in terms of

inhibiting spoilage by providing an alternative and eco-friendly way of prevention. It boosts the

precaution and prevention of mango spoilage especially at times of harvesting.

Materials and Methods

Materials

The materials such as 12 mangoes, 1 sterile zip lock plastic, 6 pair of sterile gloves, 200

g of potatoes, 20 g of dextrose powder, 20 g of gelatin (gulaman), 3 strainers, knife, cotton, 3

papers, 12 cotton swab, 1 sterile container, aluminum foil, rubber bands, 66g oregano, 9 of 0.7

mm filter paper discs were purchased from different shops of wet markets around Sta. Elena,

Marikina City. While 1 liter of distilled water, 6 beakers, 1 Erlenmeyer flask, kitchen stove,

100 mL of distilled water, 3 stirring rod, 4 cork stopper, 46 mL of distilled water, 1 mechanical

pipette with 5 sterile tips, 4 test tubes, 6 petri dishes, incubator, binocular microscope, pressure

cooker, 1 blender, stock pot, and digital weighing scale were borrowed from the Sta. Elena

High School and Marikina Clean Food and Water Laboratory.

Collecting and Storing of Deteriorated Mangoes

The researchers collected 12 mangoes (4 set ups with 3 mangoes each) by wearing 6 pair

of sterile gloves in wet markets around Sta. Elena, Marikina City. The ripened mango fruits that

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have been selected are moderately diminished in quality and about to deteriorate. Mangoes were

stored for 16 days to enable it to naturally spoil. They stored the 12 mangoes in a single sterile

zip lock plastic at a room temperature. Sterile gloves were used for storing 12 mangoes to insure

that no other microorganisms infected the samples. After the storage, molds were developed and

increased in number.

Making Oregano Extract

66 g of oregano plant were washed with distilled water and chopped into small pieces using

knife. Chopped oregano were steamed to get the extract easily using stock pot and placed in a

sterile container. Oregano extract was squeezed manually using hands with sterile gloves

Different Concentrations of Oregano Extract

Different concentrations of 10 ml oregano extract were prepared: 30 % has 3 ml oregano

extract and 7 ml distilled water, 60 % has 6 ml oregano extract and 4 ml distilled water. 90 % has

9 ml oregano extract and 1 mil distilled water.

Producing Potato Dextrose Agar

200g of potatoes were peeled and cut into smaller pieces using knife. After being peeled and

chopped, it was placed in a pressure cooker together with 1 liter of distilled water and boiled it

for 20 minutes in a stove. Boiled potatoes were filtered using strainer to separate the extract from

the undissolved potato bits and placed it in a beaker. 100 mL of distilled water was added to

make 1 liter solution. The solution was boiled with 20 grams of gelatin (gulaman) bits until the

gelatin dissolves. Then 20 g of dextrose powder was added. Solution was stirred using a stirring

rod. I was placed in beakers and covered each mouth with cotton, paper, and aluminum foil and

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secured with rubber bands. The researchers put the container in a pressure cooker with 1 liter of

water and heated for 35 minutes for the sterilization. Afterward, it was cooled for 15 minutes.

Then poured in 3 Petri dishes then marked with quadrants and became solid in 30 minutes.

Obtaining Molds in mangoes

The deteriorated 12 mangoes (4 set ups with 3 mangoes each) were shredded, and the

texture became viscous using blender. The shredded bits were separated with a strainer or

filtration. The 25 g of viscous molds was measured using digital weighing scale and placed in

Erlenmeyer flask. The 225 mL of distilled water was added in the Erlenmeyer flask with 25 g of

viscous molds. The diluted sample was covered and mixed for 1-2 minutes.

Tests and Experimentations

A. Dilution

The researchers prepared a 9 mL of distilled water in 1 test tube. They carried 1 mL of

diluted sample marked 10^ 0 to the first test tube of distilled water marked 10^-1 using

mechanical pipette. Using cork stopper, the first test tube was covered and mixed thoroughly for

about 1-2 minutes to make the sample diluted.

B. Spread plate technique

12 sterile cotton swab were used for getting the diluted sample (10^-1) in test

tube. Dipped the first cotton swab in the diluted sample (10^-1) in test tube and spread to the first

Petri dish with potato dextrose agar (media). The sample was spread on the surface of the media

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evenly by rotating the Petri dish marked with quadrant underneath at the same time allowing the

growth of molds. Same process was done in the second Petri dish up to the third Petri dish

marked with potato dextrose agar (media) using 11 sterile cotton swab.

C. Zone of Inhibition

The extract of the Oregano (Origanum Vulgare) that has antimicrobial properties was

used for the inhibition of different molds specifically the molds. The proponents provided 9 disc-

shaped filter paper for the zone of inhibition with a diameter of 0.7 mm which were placed in

different concentration of oregano extract, the 30% of the oregano, 3 mL oregano extract mixed

in 7 mL of distilled water, 6 mL extract and 4 mL distilled water in 60% oregano extract, and 9

mL of oregano extract with 1 mL distilled water for the 90% extract. Disc-shaped filter paper

were placed in each quadrant of the 3 Petri dishes except for the 0% (controlled group) using

tweezers and covered each samples. Lastly, the researchers placed the three petri dishes in the

incubator - 30°C for 4 days to get the data needed.

D. Microscopic Observation of Mold

The varieties of molds in a potato dextrose agar were placed on the binocular microscope.

The researchers rotated the ocular lenses to no. 64 lines up with the white line below the rotating

part of the lens. The proponents checked if the condenser lens was below the stage to focus the

light onto the specimen. The sample was placed on the stage with stage clips. By using a higher

power lens/magnification of 40 times, the researchers were able to set a closer look on the

different types of mold. Through the use of images in different studies, they were able to identify

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the type of molds present in the Mango (Mangifera Indica), these are the Aspergillus niger,

Aspergillus flavus, and Penicillium spp.

Data Analysis

After obtaining the data from the zone of inhibition, the following were used to

determine the effect of oregano extract:

1. Mean and standard deviation was used to evaluate the capability of oregano extract to

inhibitor the proliferation of molds in mango.

2. Pearson R was used to determine the correlation between the increasing concentration of

oregano extract and the zone inhibition.

3. Tables and graphs were used to see the trends and analyze data.

Results

This chapter presents data gathered and collected after conducting thorough

methodological processes. The data collected by the researchers were then summarized in terms

of tables and graphs.

Table 1 – Different Concentration of Oregano Extract and the Zone of Inhibition of Mango
Mold

Concetration of Oregano Extract in (10 ml) Solution

30% 60% 90%
Inhibitioni

13 15 18
Zone of

n (mm)

9 12 16
12 14 20
Mean 11.33 13.67 18.00
SD 2.08 1.53 2.00
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 Table 1 shows the zone of inhibition of every sample in different concentration of

oregano extract. Base from the mean, significant increase the zone of inhibition can be seen in

the samples as the concentration of oregano extract increases.

Table 2 – Different Concentration of Oregano Extract and the Mean of Zone of Inhibition
of Mango Mold

Concentration of Zone of
Oregano Extract Inhibition (mm)
in 10 ml Solution
0% 0
30% 11.33
60% 13.67
90% 18.00

20.00
18.00
16.00
Zone of Inhibition (mm)

14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
0 20 40 60 80 100
Concentration of Oregano Extract in Percentage

Graph 1 – Relationship between the concentration of oregano extract and zone of

inhibition

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 Graph 1 shows the mean of zone of inhibition and its correlation with the different

concentration of oregano extract. The graph also suggests perfect correlation between these

variables. It can be seen that as the concentration of oregano extract increases the zone of

inhibition also increases. The value of the correlation coefficient using Pearson R is 0.99

Discussion

The purpose of the study is to inhibit the proliferation of molds in mango using oregano

extract which will be observed based on zone of inhibition. The procedure involved the use of

spread plate method in cultivating the molds of mango in the potato dextrose agar and the

inhibitory effect of oregano extract observed through zone of inhibition. The results obtained

from the zone of inhibition of molds using oregano extract with increasing concentration imply

the significant correlation of different concentrations to the zone of inhibition. This means that

the increasing concentration of oregano extract is directly proportional to zone of inhibition

measured by its diameter.

The mean of molds without oregano extract was measured to be 0 mm while the mean of

molds with oregano extract was measured as 10.75 mm which suggests that adding oregano

extract made the zone of inhibition present. Graph no. 1 shows the capability of oregano extract

as an inhibitor in inhibiting the growth of molds.

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 Inhibition of molds obtained a mean of 11.33 mm in 30%, 13.67 mm in 60%, and 18 mm

in 90 % concentration of oregano extract. Thus, the result from the zone of inhibition indicated

that increasing concentration of oregano extract expands the diameter of zone of inhibition in

molds. This suggests that the third set up with 90% concentration of oregano extract has the

largest diameter in zone of inhibition.

The results correspond to an earlier study that the antimicrobial properties of plant

essential oils against human pathogens by zone of inhibition was performed. The oil of oregano

(Origanum Vulgare) was efficient at inhibiting the Aspergillus niger, Aspergillus flavus,

Microsporum gypseum, Microsporum canis, Arthroderma cajetani, Trichophyton violaceum,

Trichophyton mentagrophytes, Epidermophyton floccosum, T. rubrum, and Trichophyton

tonsurans (Bozin B, et al.,2006).

Conclusion

The zone of inhibition observed stated that oregano extract is an effective inhibitor of

molds in mango. It also implies that increasing the concentration of oregano extract widen the

zone of inhibition of molds. Thus, oregano extract can indeed inhibit molds causing spoilage and

deterioration of mangoes.

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 Recommendations

The researchers recommend to use oregano oil, rather than oregano extract to obtain a

much visible and effective zone of inhibition. There could be a better way for culturing

microorganisms, one that does not involve too much days. To obtain a better culturing for

microorganisms, buying a commercial product of agar is advised rather than making an

alternative. The researchers propose to use diverse antibiotic and antibacterial plants to identify

which is a better inhibitor of microorganisms. To make this study vast, other proponents could

also use different samples of foods that can be inhibited by plants from spoiling and

deterioration. The researchers also suggest identifying the bacterial properties of deteriorated

foods, not only the molds present. In identifying the capacity and capability of antimicrobial

plants, a different way of identification can be used instead of using the zone of inhibition.

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