Mycofloral of Smoke-Dried Fishes Sold in Uyo, Eastern Nigeria

World Journal of Agricultural Sciences 4 (3): 346-350, 2008
ISSN 1817-3047
© IDOSI Publications, 2008
Mycofloral of Smoke-Dried Fishes Sold in Uyo, Eastern Nigeria
Bukola. C. Adebayo-Tayo,
1


Abiodun. A. Onilude and
2


Ukpe Grace Patrick
1
Department of Microbiology, Faculty of Science University of Uyo, Uyo Akwa Ibom State, Nigeria
1
Department of Botany and Microbiology, University of Ibadan, Ibadan, Oyo State, Nigeria
2
Abstract: This study was aimed to estimate the mycoflora and aflatoxin contamination of smoked dried fishes
of Stock (Gadus morhua), Skip jack tuna (Katsuworus pelamis), Croaker (Pseudotolithus typhus), Sting ray
(Dasyatis margarita), Cat (Arius hendeloti), Bonga (Ethalmosa fimbriota), Ribban fish (Triuchurius
trichurius), Stark (Carchanas faunis), Thread fin (Pentanemis qumquarius), Sole (Cynoglossus browni), Spade
(Drepane africana) in Uyo, eastern Nigeria. Fifty-five smoke-dried fishes samples sold at three different markets
in Uyo town, located at main markets, Itam and Akpan Adem in Uyo, Akwa lbom state, Nigeria were heavily
contaminated with moulds. Twelve different fungi were found associated with the smoked dried fishes
samples sold in the three different markets. The associated fungi were Aspergillus flavus, A. tereus.
Aspergillus fumigatus, Absidia sp., Rhizopus sp., A. niger, Mucor sp., Cladosporium sp, Penicillium
Italiculum, Penicillium viridatus Candida tropicalis and Fusarium moniliformis, Aspergilus flavus and
A. tereus had the highest rate of occurrence among the isolated fungi. Aflatoxin B and G
1
were found associated
1
with the samples. The Aflatoxin B
g
a
1 and G

concentrations in the sample were between 1.5
1
– 8.1 µg/kg and
1.8j- 4.5

aµg/kg respectively. The fungal counts were between 3.0 x 10








- 4.8 x 10
2

cfu /g. The moisture conten
4
t
and the pH were between 22.7 – 27.6% and 3.0 – 6.0 respectively. In conclusion smoked dried fishes stored for
sale in Uyo markets were heavily contaminated with aflatoxigenic fungi.
Key words: Smoked dried fish % Aflatoxin % Aspergillus flavus % Mycoflora
INTRODUCTION
camps and fish processing centuries in traditional
smoking kilns of clay, cement blocks, drums or iron sheets
Fish supplies a good balance of protein, vitamins and
[5]. This result in a very short shelf life and low market
minerals. It has a relatively 10% calories content hence its
value as well as inability to withstand handling and
role in nutrition is recognized [1].
transportation by retailers [1].
Fish and fish products constitute more than 60% of
For long, fungi were regarded as causing only
the total protein intake in adults especially in the rural
anesthetics spoilage of food. But during 1966 when the
areas [2]. They are widely accepted on the menu card and
famous “Turkey X” diseases killed 10,000 turkey poultry
form a much-cherished delicacy that cuts across socio-
in Great Britain, Western world became aware that
economic, age, religious and educational barriers [2]. Fish
common spoilage molds could produce significant of
flesh is one of the best sources of protein. Its flesh is
toxigenic fungi and potentially toxic compounds have
tender due to bundles of muscle fibers, which are held
been discovered. Aflatoxins, a group of toxic metabolic
together by fibrous material when heated [3]. It is better
produced by certain Aspergillus species have been found
digested than beef or other types of protein.
to be carcinogenic tetratogenic and mutagenic to several
In Nigeria, fish is eaten fresh, preserved or processed.
species of experiment animals [6-8]. Aflatoxin occurs in a
The percentage composition of the different methods of
variety of crops and animal product. The conditions that
fish disposed for consumption in the artisanal sector
contribute to fungal growth and production of aflatoxins
according to Tobor [4] are as follows live fish 7%, fresh
are a hot and humid climate, moisture content of 16% and
fish 27%, smoke dried 45%, sun dried 20% salted and sun
above favorable substrate characteristics and factors that
dried 10%. Smoke drying methods used in Nigeria requires
decrease the host’s immunity such as insect damage [9].
low capital, investment and it is conducted in fishermen
Aflatoxins have a high melting point i.e. 250°C. It has been
Corresponding Author: Dr. Bukola. C. Adebayo-Tayo, Department of Microbiology, Faculty of Science University of Uyo,
Uyo Akwa Ibom State, Nigeria
346

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World J. Agric. Sci., 4 (3): 346-350, 2008
proved that food items do carry residue of the toxin.
The electrode was rinsed and immersed into the fish
Thus, it’s certain that human beings are exposed to
slurry. The pH reading were then recorded. Determination
aflatoxins through contaminated food items among which
were done in triplicates and the mean value were obtained.
fish is an important component [10].
This research was embarked upon to investigate the
Aflatoxins Detection in Smoke Dried Fish’s Samples:
mycoflora associated with smoke dried fishes in Uyo,
Aflatoxins were extracted from the samples according to
eastern Nigeria and the presence of aflatoxins in the
the method of Seitz and Mohr [15]. Ten grams of the
smoke-dried fishes.
fishes samples obtained from each of the markets were
weighed aseptically and macerated using a Warring
MATERIALS AND METHODS
blender and were extracted with chloroform and
concentrated. Thin layer chromatography of aflatoxin and
Sample Collection: Smoke dried fishes (Stock, Skip jack
samples extracted were performed on silica gel DG 254. Of
tuna, Croaker, Sting ray, Cat, Bonga, Ribban, Stark,
the extracted samples 5, 10 and 15 µl were spotted on
Thread fin, Sole and Spade) were randomly sampled and
three different points on a ruled base line of the TLC
purchased from three different marketing sites located at
plates. Also 5, 10 and 15 µl of the aflatoxin standard were
main markets, Itam and Akpan Adem in Uyo town Akwa
spotted on another three points near the previous sample
lbom State, Nigeria. Fifty-five samples of related species
extract spotted points. The plates were developed first
were grouped together to make eleven composite samples,
with diethyl ether and then with chloroform: acetone
they were subsequently kept in sterile polyethylene bags,
(9:1v/v). Aflatoxin was identified on the basis of co-
migration with aflatoxin standards (Fluka) and by their
which were used for analyses.
characteristic fluorescent color under long Ultra Violet
(UV) illumination was at 360 nm. The fluorescent spots of
Isolation of Fungi: Attempts to isolate fungi from the
aflatoxins were scraped off the TLC and eluted by
smoke-dried fish’s samples were made aseptically on
chloroform: methanol (9:1 v/v). The solvent was
Saboraud dextrose agar. Ten grams of the fishes samples
evaporated under nitrogen to dryness and the residue
obtained from each of the markets were weighed
was dissolved in methanol. The concentration of
aseptically and macerated in 90 ml sterile watery agar
aflatoxins (B and
1
G1) in solution was determined by


(0.2%) using a Warring blender. From this, subsequent
measuring its absorbance at 360 nm then calculated
tenfold dilution was made up to 10-5 [11]. One milliliter of
according to the method of Masri et al., [16].
each dilution was dispensed in triplicate in sterile Petri
dishes. Molten Saboraud dextrose agar to which penicillin
Confirmatory Tests for Aflatoxin: Three different
and streptomycin had been incorporated were added to
derivatives were prepared by treating portions of the
the Petri dishes, which were gently rotated to ensure even
isolated toxin or the aflatoxin standard with formic acid
dispersion. The plates were allowed to solidify and were
thionyl chloride, acetic acid-thionyl chloride and tri-
incubated at 28±1°C for 3-5 days. All observed colonies
fluoroacetic acid. The test was then continuing according
were subculture to obtain pure cultures which were
to the method of Stoloff and Friedman [17].
subsequently isolated and identified using morphological
characteristics, spore formation, the production of fruiting
Statistical Analysis: Duncan multiple range test was
bodies and biochemical reactions [12-14] and by compares
used to compare significant differences between the
with already identified cultures, which were obtained
means [18].
from the plant pathology laboratory of the Institute
of Agricultural Research and Training, Obafemi
RESULTS AND DISCUSSION
Awolowo University Moor Plantation, Ibadan, Nigeria.
The moisture content was determined by oven drying
Results obtained from this study showed that
at 105°C for 4 /2h.
1
Aspergillus flavus, Aspergillus tereus, A.fumigatus,
Absidia sp., Rhizopus sp., Aspergillus niger, Mucor sp.
Determination of pH: Two grams each of the macerated
Cladosporum sp., Penicillium italicum, Penicilium
fish’s samples were weighed in triplicates. Water was
viridatus, Candida tropicalis and Fusarium moniliformis
added and mixed thoroughly to make a fish slurry. The pH
were found to be associated with smoked dried fishes
readings were taken using digital pH meter equipped with
sold in different market in Uyo. As stated above,
a glass electrode (digital thermo pH meter mod B-E105).
Aspergillus flavus and Aspergillus tereus, A. fumigatus
347

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World J. Agric. Sci., 4 (3): 346-350, 2008
Table 1: Fungi count, pH and moisture content (%) in different fish samples

20
Samples
Fungi count
pH
Moisture content (%)
3
15
A
4.8 x 10
3.0
25.2
B
5.8 x 103
5.0
25.7
10
C
1.72 x 104
3.0
26.6
D
6.4 x 103
5.0
26.3
5
E
3.0 x 102
6.0
22.7
0
3

F
2.3 x 10
3.0
24.9

e
s

r
s
s
s





m
m
m
v
u
e
t
u
u
u

s
p

s
p
l
i
s

s
p
l
i
s

i
g
a
4
r
e
c
a
i
a
i
a
a
m
t
u
i
f
o
G
4.2 x 10
6.0
27.6
.
n
d
u
l
i
c
u
i
c
a
n
.

f
l
a
i
g
A
.
t
e
r
a
i
e
m
n
p
o
A
m
A
h
l
o
s
i
d
c
b
o
e
r
i
c
a

m
2
.
f
u
i
p
A
r

h
.

i
t
a
s
p
P

t
r
o
m
H
9.0 x 10
6.0
24.3
A
.
o
.

v
a
A
D
c
o
o
P
u
d
i
d
r
u
d
M
l
a
s
a
4
C
n
a
u
I
8.4 x 10
5.0
27.2
C
F
J
2.8 x 104
4.0
27.3
Fig. 1: The rate of occurrence (%) of fungi associated
K
7.6 x 103
5.0
26.1
with marketed smoked dried fish samples Fungi
Keys:
associated with smoked dried fish samples
A: Stock fish (Gadus morhua)
B: Skipjack tuna (Katsuworus pelamis)
were the dominant mycoflora in decreasing sequential
C: Croaker (Pseudotolithus typhus)
order. Adebayo-Tayo et al., [19] reported similar result in
D: Sting - ray (Dasyatis margarita)
E: Catfish (Arius hendeloti)
marketed bush mango seeds (Irvingia spp.) stored for
F: Bonga fish (Ethalmosa fimbriota)
sale in Uyo. Penicillium viridatus, Candida tropicalis
G: Ribban fish (Triuchurius trichurius)
and Fusarium moniliformis occurred less frequently
H: Stark (Carchanas faunis)
(fig 1). The presence of A. flavus in the samples might
I: Thread - fin (Pentanemis qumquarius)
probably make its consumption hazardous to health.
J: Sole (Cynoglossus browni)
According to Akande and Tobor [1], in artisanal fishery,
K: Spade - fish (Drepane africana)
freshly caught fish are covered with damp sacks and at
Table 2: Aflatoxin B1 and G

1 concentrations in the samples
times, they are mixed with wet grass or water weeds to
Samples
AF B
-1

-1
1µgkg
AF G
1µgkg
reduce the temperature. Fish treated this way is prone to
A
4.750c
3.550
c
contamination with microorganisms such as bacteria and
B
3.55d
3.050
d
fungi. This indicates that spoilage of fish starts right from
C
2.100i
2.8200e
the aquatic ecosystem. Handling fishes are also prone to
D
3.005e
2.515
g
g
j
microbial attack especially in artisanal fishery due to
E
1.505
1.8100
F
2.5150g
2.205 h
unhygienic methods of reducing temperature. During the
G
3.0005e
3.505
c
smoke drying period, smoking kilns used in artisanal
H
2.205h
2.000
I
fishery and the overloading of the fishes on the trays
I
2.805f
2.715
f
leads to improper processing which in turn encourages
J
7.525b
3.710
b
fungal attack [5]. During storage of smoked dried fish
K
8.105a
4.51
a
products, good storage practices are not adhering by
Each value represents a mean of three replicates. Means followed by the same
wholesaler hence stores are not well ventilated and pest
letter are not significantly different by Duncan’s multiple range tests.
can easily gain access into the stores. The environment in
which fishes are displayed in the market is not always
content while specimen H had the highest moisture
hygienic and this is another avenue for microbial
content as shown in Table 1.
contamination. Very often, retailers display the smoke-
The spots from the extracted smoke dried fish’s
dried fish samples in open trays beside the gutter on
samples and the standard aflatoxin fluorescence produced
refuse heaps, this also encourages fungi attack and
bluish and greenish sport. Sharma [20] reported that the
subsequent production of toxins. This is in agrement
two major metabolites of Aspergillus sp. called aflatoxins
with the report of Akande and Tobor [1]. The result also
were designated B1 and

G
1 because they fluoresce
revealed that the average mould count ranged from
blue (B ) and green (G
1
) when exposed to long-wave



1
3.0×102-8.4×104 cfu/g (Table1). The microbial levels
ultraviolet light.
obtained in this report which is 104 could be considered
Aflatoxin was detected in all of the samples. The
hazardous to consumers because of the possibility of the
concentration of aflatoxin B1 and G

ranged betwee
1
n
presence of enterotoxigenic strains. The pH ranged
1.505 -8.105
g
µg/kg and 1.810
a


-4.51
j
µg/kg
a
respectively. As
between 3.0 – 6.0 and also the moisture content ranged
shown in Table 2 above samples E had the lowest AFB1
from 22.7% - 27.6%. Specimen E had the lowest moisture
and AFG concentration while sample K had the highes
1
t
348

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World J. Agric. Sci., 4 (3): 346-350, 2008
AFB
1
and AFG concentration. This indicate that the
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1
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