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IDOSR JOURNAL OF APPLIED SCIENCES 4(1) 86-93, 2019.

Isolation and Identification of Microorganisms Involved in The Spoilage of

Banana Fruit (Musa acuminata) Sold in Some Selected Markets in Eastern
Nigeria
Chibuzor C.A., Ugwuanyi R.C and Ogbonna O.A.
Department of Science Laboratory Technology, Institute of Management and Technology,
Enugu, Nigeria.

ABSTRACT
This project work was carried out to isolate and identify the microorganism involved in the
spoilage of banana fruit (Musa acuminata) sold in some selected markets in Eastern Nigeria.
Microorganisms associated with spoilage of banana (Musa acuminata) were studied using
standard microbiological methods. The serial dilution method was employed using nutrient
agar, MacConkey agar, CLED agar and Sabouraud dextrose agar. A total number of 10
samples of spoilt banana fruits(Musa acuminata) were examined by culturing them on
nutrient agar, CLED agar and MacConkey agar for bacteria and sabouraud agar for fungi.
The bacterial isolates were Escherichia coli (40.7%) which had the highest occurrence,
pseudomonas spp.(23.3), Staphylococcus aureus (18.7%) and Enterobacter spp. (17.3%) which
had the least occurrence. The fungi isolates obtained were Aspergillus niger, Aspergillus
flavus and Fusarium spp. The fungal and bacterial loads were high enough to cause food
spoilage or food infection. The presence of the isolates may be due to improper hygienic
practices from the point of harvesting, transportation and storage by handlers. Above all,
the high moisture content of banana fruit (Musa acuminata) makes it highly perishable as
its supports the growth of microorganisms.

Keywords: Microorganisms, Spoilage and Banana (Musa acuminate)

INTRODUCTION
Fruits are very important and have dietary A fruit is the edible part of a mature ovary
and nutritional qualities. Consumption of of a flowering plant. It is usually eaten
fruit products has dramatically increased raw. Fruits could also be described as the
by more than 30% during the past few succulent or fleshy covering of a nut
decades [1]. During the period 1870 - which is pulpy, often juice in character.
2004, US per capita consumption of fruits As they were developed from the flower
increased by 19.9%, to 694.3 pounds per of a plant, they consist of ripened seed or
capita per year. Fresh fruit and vegetable seeds with some tissues attached [4].
consumption increased by 25.8 and Fruits play a vital role in human nutrition
32.6%, respectively, and far exceeded the by supplying the necessary growth
increases observed for processed fruit factors such as vitamins and essential
products. It is also estimated that about minerals in human daily diet and that can
20% of all fruits produced is lost each help to keep a good and normal health.
year due to spoilage [2].
[3] Reports those 20 new human fungal Fruits are widely distributed in nature.
pathogens are documented each year. One of the limiting factors that influence
Most microorganisms that are initially the fruits economic value is the relatively
observed on whole fruit surface are soil short shelf-life period caused by
inhabitants. Vectors for disseminating pathogens attacked. It is estimated that
these microbes include soil particles, about 20-25 of the harvested fruits are
airborne spores, and irrigation water. decayed by pathogens during post-

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harvest handling even in developed lead to develop inedible, undesirable
countries [5]. quality and soft rot spoilage. In
Increasing interest in medicinal herbs has developing countries, post-harvest losses
increased scientific scrutiny of their are often more severe due to inadequate
therapeutic potentials and safety thereby storage and transportation facilities.
providing physicians with data to help Fungal fruits infection may occur during
patients make wise decisions about their the growing season, harvesting, handling,
use [6]. Fruits, apart from being taken as transport and post-harvest storage and
food also have some medicinal marketing conditions, or after purchasing
importance. The latex from the trunk of by the consumer. Fruits contain high
the pawpaw tree is applied externally to levels of sugars and nutrients element
speed the healing of wounds, ulcers, boils and their low pH values make them
and warts. The seed is also used to expel particularly desirable to fungal decayed
worm and the flower may be taken in an [10]. Studies by [11] had shown that fungi
infusion to induce menstruation. In the can survive and/or grow on fresh produce
southern part of Nigeria, fruit such as and that the nutrient content
pawpaw production has improved the diet (carbohydrate, protein and fat) of fresh
of the local people, whose diet generally produce support pathogens.
consisted of starch staples lacking
essential vitamin and minerals [7]. These Fruits are however, affected by a wide
fruits were usually displayed on benches array of microorganisms causing its
and in baskets for prospective customers decay. These microorganisms, under the
in the open markets until sold, thereby influence of environmental factors, pose a
exposing them to further microbial serious threat to fruits production.
infection beside those associated with the Spoilage refers to any change in the
fruit, surface and those from adjacent condition of food in which the food
infected fruits. becomes less palatable, or even toxic;
these changes may be accompanied by
Banana is an edible fruit, botanically a alterations in taste, smell, appearance or
berry, produced by several kinds of large texture [12]. Spoilage fungi that typically
herbaceous flowering plants in the genus produce more diverse and greater
Musa. In some countries, bananas used amounts of extracellular depolymerases
for cooking may be called plantains. The successfully attack and spoil both fruits
fruit is variable in size, colour and and vegetables [13]. Fungi in particular
firmness, but is usually elongated and produce an abundance of extracellular
curved, with soft flesh in starch covered pectinases and hemicellulases that are
with a rind which may be green, yellow, important factors for fungal spoilage.
red, when ripe. Pawpaw fruit can be Some spoilage microbes are capable of
freshly eaten or cooked. It can also be colonizing and creating lesions on
used in the preparation of jellies, juice healthy, undamaged plant tissue [14].
and jams. It has great application in the
preparation of fruit salad and desserts. Spoilage microorganisms can be
Orange juice is made from fresh healthy introduced to the crop on the seed itself,
oranges. Sweet orange oil is a by-product during crop growth in the field, during
of the juice industry produced by harvesting and post-harvesting handling,
pressing the peal [8]. or during storage and distribution. Those
The primary cell wall of fruit is composed same types of soil-borne spoilage
of approximately 10% proteins and 90% microbes that occur on produce are the
polysaccharides, which can be divided same spoilage microorganisms that are
into three groups: cellulose, present on harvesting equipment, on
hemicelluloses and pectin [9], Numerous handling equipment, in the packing
cell wall degrading enzymes can be house, in the storage facility and on food
secreted by pathogens to breach and use contact surfaces throughout the
the plant cell walls as nutrient sources distribution chain. Therefore, early
that reduced post-harvest life and finally intervention measures during crop
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development and harvesting through the this fruit demands attention. Over the
use of good agricultural practices (GAP) years, there has been an increase in the
will provide dramatic reductions in yield need to identify and isolate the fungi
loss due to spoilage at all subsequent associated with their spoilage. The aim of
steps in the food-to-fork continuum [15]; this study was to isolate and identify the
[13]. Although available literatures reveal fungi that are associated with the spoilage
that the importance of fruit is increasing of banana fruit sold in some selected
daily, the incidence of microbial attack on markets in Eastern Nigeria.
AIM OF THE STUDY
The aim of this study is to isolate and the spoilage of banana fruit (Musa
identify the microorganisms involved in acuminata).

MATERIALS AND METHODS

Materials - 48 hours. MacConkey agar and nutrient
The materials used in this study were of agar was used for bacteria isolation while
analytical standards SDA was used for fungi isolation. Total
viable aerobic bacteria count was
Methods performed in nutrient agar. At the end of
Collection of Sample the incubation periods colonies were
The banana fruit was purchased from expressed as colony forming unit of the
Ogbete Main Market Enugu State Nigeria. suspension. Desired colonies were sub-
These samples were placed in separate cultured into fresh agar plates aseptically
sterile plastic bags and transported to the to obtain pure cultures of the isolates.
laboratory for microbial analysis. Pure isolates of the resulting growth were
then stored at 40°C.
Preparation of Culture Media
The media (Nutrient, MacConkey, Biochemical Tests carried for
Sabouraud Dextrose Agar and CLED) for Identification of Bacteria Isolates
culturing were prepared according to the Catalase Test
manufacturer's directives and autoclaved A smear of a small portion of a colony
at 121° C for 15 minutes for 15 Ibs under test was placed into a tube
pressure. The media were allowed to cool containing about 2 ml of hydrogen
for 45 degrees, then swirled well before peroxide. Catalase positive strains caused
aseptically poured into Petri-dishes and effervescence (air bubbles) while catalase
allowed to solidify. negative does not.

Sample Preparation Coagulate Test

About one grams (Ig) of banana fruit was A drop of normal saline was placed on a
weighed out using a mechanical weighing clean slide. About one or two colonies of
balance, homogenized into 90 ml of the test organism were picked with a
sterile distilled water using a sterile sterile loop and emulsified in the drop of
blender. Ten fold serial dilutions of the saline to form a smooth milky
homogenates were made using sterile suspension. The inoculating wire-loop
pipettes. was dropped into undiluted plasma
obtained by centrifuging human blood to
Laboratory Isolation of Bacteria which sodium oxalate an anticoagulant,
Organism and Fungi Associated with has been added to a concentration of 0.2 -
Pawpaw 0.3 percent. Coarse clumping become
From the 10-fold dilutions of the visible to the naked eye within 5-10
homogenates 0.lml of 10-2, 10-3 and 10-4 seconds indicated a positive result while
dilutions of the homogenates was plated no reaction indicated negative result.
method. The plates were swirled clock
wisely and anticlockwise, allowed to
solidify and then incubated at 37°C for 24
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Indole Test medium. The tubes were pluged with non-
The peptone water medium was absorbent cotton-wool and sealed with
inoculated and incubated for 48 hours at aluminum foil before being sterilized with
37°C. After 48 hours of incubation, 3 autoclave at 105°C for 15 minutes at 15Ib
drops of Kovac's reagent was added and pressure. After sterilization, each tube
was shook very well and allowed to stay were inoculated with specific colony and
for 15 minutes (in each tube). The red incubated for 24 hours at 37°C and
ring on the surface of the peptone water unincubated tubes serve as control.
indicates positive result, while yellow ring
indicates negative result. Acid production was indicated by a
change in colour from orange to yellow
colour, indicating that acid has been
Sugar Fermentation Test produced and gas production was
This test was carried out using a media indicated by the presence of air spaces
called triple sugar iron agar, containing between the Durham tubes and the
dextrose (simple sugar), lactose and medium used presence of air bubbles also
sucrose. The medium was prepared indicated presence of gas interring that
according to the manufacturer's directive gas has been produced,
which is to dissolve 65gm into 100ml of
distilled water, after which 10ml of the Note
prepared medium was added into test After solidification, the plates were
tubes, 3 drops of phenol red was added. inverted and incubated for 24 hours at
Durham's tubes were inserted in an 37°C. After incubation, the grown colonies
inverted position (for detection of gas were observed and there morphologies
production) making sure it touches the were recorded.

RESULTS
Table 1: Plate Count of Viable Bacterial Organisms Isolated from Banana
Sample Number Mean Bacterial Count per ml
Banana
1 70
2 65
3 55
4 45
5 90
6 40
7 76
8 30
9 88
10 98

Average 65.7

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Table 2: Standard Plate of Different Colonial Form Isolated from Banana

Sample Code of Colonial Forms
Number Banana
A B C D
1 30 15 16 9
2 20 7 10 28
3 15 20 2 18
4 20 7 10 8
5 50 25 9 6
6 11 12 10 7
7 40 17 10 9
8 10 5 9 6
9 16 27 40 5
,0 52 20 8 18
Average 26.4 15,5 12.4 11.4

Key:
A = Escherichia coli
B = Pseudomonas spp
C = Staphylococcus spp
D = Enterobacter spp

Table 3: Morphological and Biochemical Characteristics of Bacterial isolates

Morphology

organisms
Coagulase

Identified
Reaction

Catalase
Glucose

Sucrose
Lactose

Indole
Gram

Rod -ve - + - + - + Escherichia coli

Rod -ve AG - AG - - + Pseudomonas spp

Cocci +ve AG AG AG + + - Staphylococcus spp
Cocci -ve AG - + - - + Enterobacter spp

Key:
-ve = Gram negative test
+ve = Gram positive test
A = Acid production during sugar fermentation
G = gas production during sugar fermentation
+ = positive
- = Negative

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Table 4: percentage Distribution and Bacterial Isolated from Spoiled fruit (Orange,
Pawpaw and Banana)

Name of Bacterial Mean Count of Bacterial organism Percentage Distribution

Isolated Banana Banana

A E. coli 26.4 40.7

B Pseudomonas spp 15.5 23.3
C Staphylococcus spp 12.4 18.7
D Enterobacter spp 11.4 17.3
Total 65.7 100

Table 5: Identification of Fungal Isolates

Morphology Microscopic Examination with Fungi Identified

Lactophenol
1. Pinkish shining smooth Simple branched aseptate hyphae Fusarium spp
in front view and pink with conida lined at the tips of
colour at the reverse each hyphae
view
2. Black colouration in Aseptate hyphae with rough head Aspergillus flavus
front and creamish in of pigment
reverse view
3. Colonies with loose Vesicles were light, yellow brown. Aspergillus niger
white to yellow Phialides growing radially along
mycelium, rapidly the periphery of vesicles. Primary
turning dark brown and phialides and secondary phialides
eventually black on the are bogh brown
development of conidia

DISCUSSION
The findings of this study showed that The fungi isolated in this study have been
Aspergillus flavus, AspergiUus niger, reported to produce secondary
Fusarium spp. were found in fruits sold in metabolites in plants tissues. These
some Market in Enugu state, Eastern secondary metabolites are potentially
Nigeria. All the three organisms isolated harmful to humans and animals. A good
were confirmed to be pathogenic on the example is Aflatoxin which has been
fruits but in varying degrees. It showed associated with cancer of the liver and
that of all the isolated fungi, Aspergillus also with acute hepatitis in humans,
niger was highly pathogenic. All the especially in the developing world.
organisms were successful taking part in Pathogenic fungi on the other hand, could
the decay and are thus confirmed as the cause infection [8]. AspergiUus spp. are
causal organism of fruit decay [12]; [13]. known to produce several toxic
Generally, spoiling fungi are considered metabolites such as malformins and they
toxigenic or pathogenic. Toxigenic fungi can produce a mycotoxin which is a very
have been isolated from spoiling fruits. important toxin worldwide because of the
During refrigeration some moulds may hazard it poses to human and animal
produce mycotoxins [11]. health thus extra care should be taken
during personnel handling of these fruits,
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such as harvesting, cleaning, sorting, microbial chances of inoculation,
packaging, transport and storage [6]. microbial food poison, food intoxication
and food spoilage hence, food processor
The bacterial isolates identified in this or seller of fruits may be
study include Escherichia coll, counterproductive by being responsible
Pseudomonas spp, Enterobacter spp and for public health hazard and loss of
Staphylococcus aureus as shown in Table revenue [4].
3. Microorganisms most commonly found
in fruits generally involve Pseudomonas Most of the organisms found in this study
and Staphylococcus aureus. Sufficient are those commonly found in soil and
moisture, abusive temperature and water. But the presence of other indicator
adequate time well ensure a continuing organism like Enterobacter spp. may be as
increase in the bacteria population. They a result of possible contamination during
are all associated with plant where they sales or unhygienic handling of the fruits.
are known to cause plant diseases of the In this study, the fungi isolated were
rot. E. coli are indicator of feacally Fusarium spp., Aspergillus flavus and
contaminated products. Therefore, food Aspergillus niger.
processors may be sources of these

CONCLUSION
This study detected the profile of sanitation of warehouses and disinfection
spoilage fungi and bacteria which caused of packaging and transit containers,
pathogenecity of some local fruits. It also proper handling of the fruit during
showed that fungi and bacteria were harvest to prevent bruises and scars or
involved in the spoilage of banana fruit. other mechanical injuries.
Mechanical injuries such as bruises or cut It is therefore important that both the
that occur during harvesting or post- farmer who harvests the fruits into bags
harvesting, grading and packing could for transportation, the marketers and
provide infection sites for spoilage consumers take necessary precaution in
pathogens. Fruit spoilage however can be preventing contamination and eating of
controlled by the following practices, contaminated fruits. This will however
washing of harvested fruit with clean or enhance reduction the risk of mycotoxins
pure water, proper cleaning and that are deleterious to human health.
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