Microbial load on smoked fish commonly traded in Ibadan, Oyo State, Nigeria

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*Corresponding E-mail: ayeloja2@gmail.com, aye_ayo@yahoo.com
JASEM ISSN 1119-8362
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J. Appl. Sci. Environ. Manage.
Vol. 22 (4) 493 – 497 April 2018
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Microbial Load on Smoked Fish commonly traded in Ibadan, Oyo State, Nigeria
1*
AYELOJA, AA;
2
GEORGE, FOA;
1
JIMOH, WA;
3
SHITTU, MO;
4
ABDULSALAMI,
S A
1
Department of Aquaculture and Fisheries, University of Ilorin, PMB 1515 Ilorin, Kwara State, Nigeria
2
Department of Aquaculture and Fisheries Management, Federal University of Agriculture, Abeokuta, Nigeria
3
Department of Fisheries Technology, Federal College of Animal Health and Production Technology Moor Plantation, Ibadan, Nigeria
4
Department of Biological Sciences, Aquaculture and Fisheries Unit, Crescent University, PMB 2104, Sapon Abeokuta, Ogun State, Nigeria
*Corresponding E-mail: ayeloja2@gmail.com, aye_ayo@yahoo.com
ABSTRACT:
The microbial load on smoked fish sold in Ibadan, Oyo State was studied. 64 of 4 different fish species
(Mackerel (Scomber scombrus), Sardine (Sardinela eba), Panla (Gadus morhua) and Cat fish (Clarias gariepinus)) were
sourced from five different market locations. The microbiological analysis was done using standard microbiological
procedures. The bacterial count of fish sampled from Ojo market was the highest (0.35±0.11
x 10
3
CFU/g) while the
bacteria count of the fish sample from Bodija market was the lowest (0.07±0.04
x 10
3
CFU/g). There was no significant
difference (p>0.05) in the microbial load of the various fish species sold within Ibadan metropolis as well as fish sampled
from various market locations in Ibadan metropolis. Bacterial isolated on smoked fish studied were: E. coli, Salmonella
spp, Klebsiella spp, Staphylococcus aureus, Aerosomonas spp, Pseudomonas spp, Vibrio spp, Serratia spp,
Chromobacterium spp, Enterobacteria spp and Shigella spp. While the following fungi were isolated from the study:
Aspergillus flavus, Penicillium spp, Fusarium oxysporum, Trrichoderma spp and Ceotrichium albidium. Fish species sold
in different markets within Ibadan metropolis is fit for human consumption. Caution should be exercised in consuming
smoked-dried fish displayed openly, reheating may be necessary to activate such micro-organisms before consumption.
DOI: https://dx.doi.org/10.4314/jasem.v22i4.9
Copyright: Copyright © 2018 Ayeloja et al. This is an open access article distributed under the Creative
Commons Attribution License (CCL), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Dates: Received: 17 March 2018; Revised: 07 April: 2018; Accepted: 09 April 2018
Keywords: bacteria, fungi, market, fish species
Consumption of fish and fish products is
recommended due to good digestibility and the high
content of polyunsaturated fatty acids. Fishes are a rich
source of protein commonly consumed as an
alternative source of protein due to the higher cost of
meat and other sources of animal protein (Oluwaniyi
and Dosumu, 2009). Fish has lower cholesterol
content when compared with meat and thus often
recommended for consumption especially among the
adult population (Harris, 1997). The marine fish is
generally cheaper and more abundant when compared
with fresh water fishes, which are relatively more
expensive in Nigeria (Oluwaniyi and Dosumu, 2009).
Fish is a highly perishable food and so, many
strategies have been developed to limit its spoilage
(Gómez-Estaca et al., 2009). According to Kumolu-
Johnson et al. (2010) various food preservation
techniques have been utilized to improve the microbial
safety and extend the shelf-life of fish in general
including freezing, chemical preservation, salting,
smoking, frying and filleting. However, smoking is the
most popular method of fish processing (Bako, 2004).
Food is considered to be microbiologically unsafe
owing to the presence of microorganisms which may
invade human body (e.g Salmonella, Escherichia coli,
Listeria monocytogenes, etc) and also those that
produce toxins ingested with a food such as
Staphylococcus aureus, Clostridium botulinum and
Bacilus cereus (Ofred, 2009). The growth of these
pathogens may not necessarily results in food
spoilage. Hence the absence of deleterious sensory
changes cannot be used as an indicator for food
microbiological safety (Border and Norton, 1997).
Consequently many outbreaks are caused by bacteria
originating in the animal/human reservoir including
Salmonella, Shigella, E. coli, and Staphylococcus
aureus, (Huss et al., 1997). Generally fish and
shellfish are common vehicles of transmitting food
borne diseases. It is known that the primary source of
cholera is feces of infected persons and transmission
of bacteria with water and food. However, at present it
has also been found that in certain areas the aquatic
environment is a natural reservoir (habitat) for Vibrio
cholerae. Microbiologists are of the opinion that the
presence of Escherichia coli in food indicates the
probability of contamination of the food products with
the sewage of human or animal origin (Ofred, 2009).
Data on risk factors for foodborne diseases indicate

Microbial Load on Smoked Fish commonly….. 494
AYELOJA, AA; GEORGE, FOA; JIMOH, WA; SHITTU, MO; ABDULSALAMI, S A
that the majority of outbreaks result from
inappropriate food handling practices (Jones and
Angulo, 2006). Incidences of food borne disease
outbreaks are been reported each year in Kenya
(Githiri et al., 2009). However, there is scarcity of
information on occurrences of pathogens on fish and
other ready to eat food in Nigeria thus the need for this
study with the objective of determining some
microorganisms that are prevalent on smoked fish sold
in markets in Ibadan Oyo State Nigeria.
MATERIALS AND METHODS
Sample Collection: 3 replicates of 4 different smoke
dried fish including Mackerel (Scomber scombrus),
Sardine (Sardinela eba), Panla (Gadus morhua) and
Cat fish (Clarias gariepinus) were purchased from 5
different markets; OJoo, Apata, Iwo-road, Bodija and
Omi-Adio, in Ibadan, Oyo state South-Western
Nigeria (totaling 60 fishes). The fish samples were
collected and kept in sterile polythene bags for
microbial analysis.
Preparation of Materials: The working tables were
swabbed with ethanol to disinfect them. All the wares
were washed and air-dried after which they were
sterilized in hot air oven at 160
o
C for 1hour. Culture
media like nutrient agar was prepared according to
manufacturers’ specifications and distilled water used
for serial dilution was sterilized in an autoclave at
121
o
C for 15minutes before use.
Preparation of agar plate: The agar plates were
prepared by first sterilizing the petri dishes. This was
done by putting the petri dishes in petri dish cans in
the hot air oven at 160
o
C for 1hour. The sterilized
plates were then left in petri dish cans until required,
the wire loop was sterilized by flaming in red-hot fire
using a spirit lamp. The agar were prepared by
dissolving 0.6g of the agar in 100ml distilled water and
the sterilize using a microwave at 121
o
C for 15min and
left to cool to 45
o
C.
Preparation of fish samples for microbiological tests:
Fish samples were collected in triplicates from the
samples bought from different markets. The fish were
minced after which serial dilution was made, total
viable count was done, colonies on the plates were
picked and sub-cultured and then identified.
Serial dilution: 10g of each fish sample were weighed
aseptically and homogenized in 90ml sterile peptone
water. Then, serial dilutions was made by mixing
1.0ml of the suspension in 9.0ml sterile peptone water
to obtain 10
-1
dilution. The dilution was then made to
10
-2
, and 10
-3
diluents, then spread-plated on plates of
nutrient agar (for total viable counts); Salmonella-
shigella agar (for Salmonella and Shigella species);
Mannitol salt agar (for Staphylococcus spp); Listeria
agar base (for Listeria monocytogenes); MacConkey
agar (for E. coli and other enteric bacteria); Robertson
cooked meat medium (for Clostridium botulinum) and
Eosin Methylene blue agar (for enteric bacteria). The
plates was triplicated and incubated at 37
o
C for
24hours. Total number of cells per gram of samples
were then estimated after counting the colonies on the
plates. Colonies on the plates were then picked and
sub-cultured on nutrient agar plates to ensure purity of
cultures. The different pure cultures were then
transferred to nutrient agar slopes and then identified.
Characterization and identification of the isolates:
Bacterial isolates were characterized using routine
microbiological procedures as described by Olutiola et
al. (1991); Fawole and Osho (1995) after which they
were identified using Bergey’s Manual of
Determinative Bacteriology. The microbiological
identification procedures used include:
Colony morphology: This involve the microscopic
evaluation of the characteristics of bacteria colonies on
the agar plates. The characteristics considered include
the shape of the colony, elevation of the colony, edge
of the colony, colony surface pigmentation and the
optical characteristics.
Cell morphology: This shall involve staining of the
isolates to show the cell shape and appearance. In this
study, gram staining method was carried out on each
isolate. This involve studying the isolates under the oil
lens immersion microscope after gram-staining. A thin
smear of each isolate was made and heat-fixed. The
heat-fixed smears was covered with crystal violet for
about 1 minute and immediately rinsed with clean
water. The smear was then flooded with iodine for 1
minute and then rinse immediately. The smear was
then decolorized for 10 – 30 sec using 95% ethyl
alcohol. Alcohol action was terminated by rinsing the
slide with clean water, then the smear was counter-
stained with safranin for 30seconds and then it was
rinsed off using clean water and after which it was
allowed to air dry. The stained slides were examined
under the microscope (with the aid of immersion oil)
for results. Gram-positive organisms appeared in
purple while gram-negative organisms appeared in
pink or red.
Motility test: This was carried out using hanging drop
method (Fawole and Osho, 1995). Here, a clean
depression slide and cover glass was used, they were
washed and rinsed to provide a grease-free slide. Then
a very small amount of Vaseline was placed near each
corner of the cover slide. Two loopful of the isolate

Microbial Load on Smoked Fish commonly….. 495
AYELOJA, AA; GEORGE, FOA; JIMOH, WA; SHITTU, MO; ABDULSALAMI, S A
were placed in contact with the cover glass with the
depression slide put over the drop of suspended
bacteria. The slide was quickly inverted and examined
under the microscope, then the motion of the
organisms were observed.
Biochemical Test: Some of the biochemical test
carried out include: catalase test, indole test, citrate
test sugar fermentation test and oxidase test.
Statistical Analysis: The results are expressed as
means and standard deviation and analysed using two
ways analysis of variance (randomized complete block
design) using SPSS version 17.0 where significant
difference (p<0.05) recorded. Duncan multiple range
test was used to separate the means.
RESULTS AND DISCUSSION
The microbial load on fish from different market
locations in Ibadan metropolis are presented in Figure
1. The highest bacteria load was recorded on fish
samples from Ojo market followed by those from
Apata market while the lowest bacterial count was
recorded on fish samples from Bodija market.
Fig 1: Microbial Load on Fish from Different Market Location in
Ibadan Oyo state
The result in table 1 show that the highest fungi count
was recorded on fish samples from Omi market (0.05±
0.07
x 10
3
CFU/g) followed by those samples from
Apata market (0.04± 0.07
x 10
3
CFU/g) while the
lowest fungi count was recorded on fish samples from
Ojo market (0.16± 0.10
x 10
3
CFU/g). The highest
bacterial load on different fish types within Ibadan
metropolis as presented on figure 2 was recorded on
Panla (Gadus morhua) with the value 0.27±0.19
x 10
3
CFU/g, followed by sardine (Sardinela eba) with
value 0.27±0.19
x 10
3
CFU/g while the lowest bacteria
count was recorded on catfish (Clarias gariepinus)
samples with value 0.14±0.10 x 10
3
CFU/g. Also,
represented in figure 2 was the fungi load on different
fish types in Ibadan metropolis. The highest fungi
count of 0.09±0.07 x 10
3
CFU/g was recorded on
sardine (Sardinela eba) followed by 0.08±0.12
a
x 10
3
CFU/g recorded
for catfish (Clarias gariepinus) and
Panla (Gadus morhua) while the lowest fungi count of
0.06±0.04 x 10
3
CFU/g was recorded Mackerel
(Scomber scombrus). However, there is no significant
difference (P>0.05) on the microbial load of the
different fish species sold in Ibadan metropolis.
Fig 2: Microbial Count on Different Fish Type in Ibadan Oyo State
Table 1: Microbial Load on Fish from Different Market
Location in Ibadan
Market TVC (Cfu/g) Fungi (Cfu/g)
Ojo 0.35±0.11
a
x 10
3
0.16± 0.10
a
x 10
3
Bodija 0.07±0.04
a
x 10
3
0.05± 0.07
a
x 10
3
Iwo-road 0.12±0.10
a
x 10
3
0.08± 0.05
a
x 10
3
Apata 0.30±0.20
a
x 10
3
0.04± 0.07
a
x 10
3
Omi 0.22±0.32
a
x 10
3
0.05± 0.07
a
x 10
3
SEM 0.04 0.02
Column mean with the same superscript are not significantly
different (p>0.05)
Table 2: Microbial count different fish species sampled
Fish spp TVC (Cfu/g) Fungi (Cfu/g)
Mackerel 0.19±0.22
a
x 10
3
0.06±0.04
a
x 10
3
Catfish 0.14±0.10
a
x 10
3
0.08±0.12
a
x 10
3
Panla 0.27±0.19
a
x 10
3
0.08±0.71
a
x 10
3
Sardine 0.25±0.20
a
x 10
3
0.09±0.07
a
x 10
3
SEM 0.04 0.02
Column mean with the same superscript are not significantly
different (P>0.05)
The result of this study (table 1) indicate that there is
no significant difference (P>0.05) on the microbial
load of fish sampled in the different market location
meaning that the fish sold in different markets in
Ibadan Oyo state have similar microbial load which
still fall within the recommended microbial limit in the
guidelines for the microbiological quality of various
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
Ojo Bodija Iwo
Road
Apata Omi
Microbial Load
Market Location
Bacteria Count
Fungi Count
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Mackerel Catfish Panla Sardine
Microbial Count
Fish Type
Bacteria Count
Fungi Count

Microbial Load on Smoked Fish commonly….. 496
AYELOJA, AA; GEORGE, FOA; JIMOH, WA; SHITTU, MO; ABDULSALAMI, S A
ready-to-eat foods by Gilbert (2000) and Taoukis et al.
(1999) that is the fish sold in the various markets in
Ibadan is fit for human consumption. The microbial
load on the various fish spp still fall within the
recommended microbial limit in the guidelines for the
microbiological quality of various ready-to-eat foods
by Gilbert (2000) and Taoukis et al. (1999). Result
obtained from the study showed that Aspergillus
flavus, Geotricium albidium, Trichoderma sp.,
Fusarum oxysporium, Penicillium spp, Aeromonas
spp, Klebsiella oxytoca, Levinae spp, Serratia
rubidaea, Proteus vulgaris, Shigella flexneri,
Enterobacter aerogenes, Vibrio cholerae, Escherichia
coli, Salmonella arizonae, Streptobacillus
monilliforms, Bacteria necrophorus, Pseudomonas
amattophillia and Chromobacterium violeceum, are
the microorganisms found to be associated with smoke
dried fishes sold in different market in Ibadan. The
presence of all these bacteria and fungi may make its
consumption hazardous to health as some of these
microorganisms have been reported by other authors
has been hazardous for human consumption. Fawole
and Osho (1995) reported that Spoilage of fish is
mainly due to the activity of psychrotrophic gram-
negative bacteria such as Shewanella putrefaciens and
Pseudomonas spp. Akande and Tobor (1999) also
reported that fish and fish products spoil by different
specific spoilage organisms (SSO) depending on the
final treatment or preservation and storage
temperature. Examples of SSO in different fish and
fish products include Pseudomonas, Shewanella
putrefaens, Photobactereum phosporeum, Aeromonas
hydrophila, and Alteromonas putrefaciens,
Vibrionaceae, Aeromodans, Moraxella,
Acinetobacter, Enterobacteriaceae; and Yeast and
molds. According to Ayeloja et al. (2011), in artisanal
fishery, freshly caught fish are covered with damp
sacks and at times they are mixed with wet grass or
water weeds to reduce the temperature. Fish treated
this way is prone to contamination with
microorganisms such as Salmonella and Aspergillus.
This indicates that spoilage of fish starts right from the
aquatic ecosystem. Processed fishes are also prone to
microbial attack especially in artisanal fishery due to
unhygienical method of processing and preservation.
During the smoke drying period, smoking kilns used
in artisanal fishery and overloading of the fishes on the
trays lead to improper processing which in turns
encourage fungal attack. During storage of smoked
dried fish product, good storage practices are not
adhered to by fish mongers hence stores are not well
ventilated and pest can easily gain access into the
stores. The environment in which fishes are displayed
in the market is not always hygienic and this is another
avenue for microbial contamination very often,
retailers’ display the smoke dried fish sample in open
trays beside the gutter or refuse heaps, this also
encourages fungi and bacteria attack and subsequent
production of toxins. This is an agreement with the
report of Ayeloja et al. (2011).
Conclusion: The study revealed that smoked-dried
fishes in Omi, Iwo Road, Bodija, Ojoo and Apata
markets in Ibadan Oyo State are contaminated with
micro-organisms. However, the microbial load still
fall within the recommended microbial limit for ready-
to-eat foods indicating that the fish sold in different
markets within Ibadan metropolis is fit for human
consumption. The microbial load on different fish spp
in Ibadan metropolis are not significantly different (p
> 0.01) from each other. It is therefore recommended
that fish stores should be well screened and ventilated
so as to avoid contamination of smoked fish products.
REFERENCES
Oluwaniyi, OO; Dosumu, OO (2009). Preliminary
Studies on the effect of processing methods on the
quality of three commonly consumed marine
fishes in Nigeria. BIOKEMISTRI. 21 (1): 1-7.
Harris, WS (1997). N-3 fatty acids and serum
lipoproteins: human studies. Amer. J. Clinical
Nutri. 65:1645-1654.
Gómez-Estaca, AJ; López, D; Lacey, MC; Gómez-
Guillén, ME; López-Caballero; Montero, P
(2009). Antimicrobial Activity of Composite
Edible Films Based on Fish Gelatin and Chitosan
Incorporated with Clove Essential Oil. Journal of
Aquatic Food Product Technology. 18 (2): 46-52.
Kumolu-Johnson, CA; Aladetohun, NF; Ndimele PE
(2010). The effects of smoking on the nutritional
qualities and shelf-life of Clarias gariepinus
(BURCHELL 1822). Afr. J. Biotechnol. 9: 73 -76.
Bako, WS (2005). The role of women in fish
processing: Handling and marketing in Kainji
Lake basin. Proceedings of 19
th
Annual
Conference of the Fisheries Society of Nigeria
(FISON), Nov. 29 – Dec. 3, 2004, Ilorin, Nigeria.
Ofred, JMM (2009) Microbiology and Spoilage Trail
in Nile Perch (Lates niloticus), Lake Victoria,
Tanzania. A thesis submitted for a partial
fulfilment of the requirement for the degree of
Masters of Science in Food Science (Seafood
Processing) Faculty of Food Science and
Nutrition, School of Health Sciences University
of Iceland Reykjavik, ICELAND. Pp94.

Microbial Load on Smoked Fish commonly….. 497
AYELOJA, AA; GEORGE, FOA; JIMOH, WA; SHITTU, MO; ABDULSALAMI, S A
Border, P and Norton, M (1997). Safer eating:
microbiological food poisoning and its
prevention. The Parliamentary Office of Science
and Technology, London.
Huss, H; Dalgaard, P and Gram, L (1997).
Microbiology of fish and fish products. In Luten
J. B., Borresen T., and Oihlenschlager J., (1997):
Seafood from producer to consummer, intergrated
approach to quality. Development in food science
38. Proceedings of the International Seafood
Conference on the occasion of the 25th
anniversary of the WEFTA, held in
Noordwijkerhout, The Netherlands, 13-16
November.
Jones, TF; Angulo, FJ (2006). Eating in Restaurants: a
risk factor for food borne disease. J. Clinical
Infection Diseases. 43: 1324 -1328.
Githiri, M; Okemo, P and Kimiywe, J (2009).
Hygienic practices and occurrence of coliforms
and Staphylococcus on food at a public hospital in
Kenya. J. Appl. Biosciences. 27: 1727 – 1731.
Olutiola, PO; Famurewa, O; Sonntag, HG (1991). An
introduction to General Microbiology: A Practical
Approach. Heidelberger Verlagsanslait
Druckerei, Heidelberg Germany. Pp112 – 175.
Fawole, MO and Osho, BA (1995). Laboratory
manual of microbiology. Shalom prints Ibadan
Nigeria. Pp25 – 30.
Centre for Food Safety Food and Environmental
Hygiene Department (CFSFEHD) (2007).
Microbiological Guidelines for Ready-to-eat
Food. Risk Assessment Section Centre for Food
Safety Food and Environmental Hygiene
Department, Hong Kong. 15pp.
Gilbert, RJ (2000). Microbiological Guidelines for
Ready-to-Eat Foods. Food watch, western
Australian food monitoring program. Pp5.
Taoukis, PS; Koustsoumsnis, K; Nychas, GJE (1999).
Modelling of spoilage microflora of boque (oops
boops) as a bases for chilled distribution
monitoring with time –temperature indicators. In
Bourgeois, C. M, Roberts T. A. (Eds.), Predictive
microbiology applied to chilled food preservation
(Proceedings of the international Symposium,
Quimper, France, June 16-18, 1997).
Refrigeration Science and Technology
Proceedings series, International Institute of
Refrigeration (IIR), Paris, France. Pp316-325.
Akande, GR and Tobor, JG (1992). Improved
utilization and increased availability of fishing
products as an effective control of aggravated
animal protein deficiency induced malnutrition in
Nigeria proceedings of the 10
th
annual conference
of the fisheries society of Nigeria. Pp18-31.
Ayeloja, AA; George, FOA; Obasa, SO; Sanni, LO;
Ajayi, AA (2011). Effects of length of delay after
slaughter (LODAS) on raw catfish Clarias
gariepinu. J. Amer. Sci. 7 (6): 508 – 512.