Distribution of aflatoxin and aflatoxigenic, and other toxigenic fungi in maize samples marketed in IBadan, Oyo state, Nigeria

Abstract

Twelve maize samples were collected from four different Local Government Areas within Ibadan Metropolis Oyo state, Nigeria and their moisture content was determined. The samples were also screened for aflatoxigenic fungi and aflatoxin contamination and also the presence of other fungi. The results of the moisture content revealed that the moisture of the maize samples ranged from between 6.4% to 10.2%. Fungal contamination studies revealed that species of Aspergillus were predominant with Aspergillus flavus having 20.8% occurrence followed by Fusarium oxysporum (15%) and Fusarim solani (10) and Penicillum fellutanum (2.5%). Results of the aflatxoin contamination revealed that seven (58%) of the total twelve maize samples were contaminated with aflatoxin B1 with sample I having the highest content of 24.1 pbb closely followed by sample H with 19.47 pbb. Five (41.66%) of the maize samples was found to contain aflatoxin B2 with sample I having the highest concentration of 7.67 pbb followed by sample 4.36 pbb and the least aflatoxin B2 content was recorded in sample B (1.46 pbb). Only two (sample B and J) of the twelve maize samples were found to contain Aflatoxin G1 at concentrations of 4.89 and 0.85 pbb respectively while sample J was the only maize sample containing aflatoxin G2 type at concentration of 10.78 pbb.

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ISSN:!1579*4377!
DISTRIBUTION OF AFLATOXIN AND AFLATOXIGENIC, AND
OTHER TOXIGENIC FUNGI IN MAIZE SAMPLES MARKETED IN
IBADAN, OYO STATE, NIGERIA
Onilude, A. A1., Wakil, S. M1., Odeniyi, A. O1., Fawole O2., *Garuba E. O1 and Ja’afaru, I. M.3
1 Microbial physiology and Biotechnology Laboratory, Department of Microbiology, University of
Ibadan, Nigeria
2 Department of Microbiology The Polytechnic Ibadan, Ibadan, Nigeria
3Department of Microbiology Federal University of Technology, Yola, Nigeria
oluwaseungaruba@live.com
ABSTRACT
Twelve maize samples were collected from four different Local Government Areas within
Ibadan Metropolis Oyo state, Nigeria and their moisture content was determined. The
samples were also screened for aflatoxigenic fungi and aflatoxin contamination and also the
presence of other fungi. The results of the moisture content revealed that the moisture of the
maize samples ranged from between 6.4% to 10.2%. Fungal contamination studies revealed
that species of Aspergillus were predominant with Aspergillus flavus having 20.8%
occurrence followed by Fusarium oxysporum (15%) and Fusarim solani (10) and Penicillum
fellutanum (2.5%). Results of the aflatxoin contamination revealed that seven (58%) of the
total twelve maize samples were contaminated with aflatoxin B1 with sample I having the
highest content of 24.1 pbb closely followed by sample H with 19.47 pbb. Five (41.66%) of
the maize samples was found to contain aflatoxin B2 with sample I having the highest
concentration of 7.67 pbb followed by sample 4.36 pbb and the least aflatoxin B2 content
was recorded in sample B (1.46 pbb). Only two (sample B and J) of the twelve maize
samples were found to contain Aflatoxin G1 at concentrations of 4.89 and 0.85 pbb
respectively while sample J was the only maize sample containing aflatoxin G2 type at
concentration of 10.78 pbb.
KEYWORDS
Maize, Aflatoxin, Aflatoxigenic, Asperillus sp. and Fusarium sp.

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INTRODUCTION
Mycotoxins are toxic secondary metabolites produced by many filamentous fungi that can
contaminate various agricultural commodities in pre-harvest, harvest and post-harvest
storage conditions (Gleen, 2007). The production of a particular mycotoxin is restricted to a
limited number of fungal species and, in some instances, may be limited to a particular strain
within a species (Huwig et al., 2001). They are structurally diverse, deriving from a number
of biosynthetic pathways and their effect upon consumption by animals and humans is
equally diverse ranging from acutely toxic to immunosuppressive or carcinogenic
(Fadahunsi et al., 2011).
Although over 300 mycotoxins have been described, relatively few are of major
concern to human and animal health. One of the most important groups of mycotoxin of
great concern is the aflatoxin. They are mainly produced by certain strains of Aspergillus
parasiticus and A. flavus and they occur in agricultural products in tropical and subtropical
regions due to the prevailing weather conditions and the practice of non-scientific method of
handling grains during harvesting, shelling and drying (Garuba et al., 2011). Aflatoxins are
divided into six major groups according to their fluorescent properties under ultraviolet light
(ca. 365 nm) and their chromatographic mobility (Pestka, 2007). Aflatoxins B1 and B2
produce a blue fluorescence while G1 and G2 a green one. There are also two metabolic
products; aflatoxin M1 and M2 which occur in the milk of lactating mammals which have
consumed aflatoxin contaminated-feed. Aflatoxin B1 is the most toxic and the most
prevalent among this family (Pestka, 2007).
The consumption of aflatoxin-contaminated feed pose a health risk to animals, and a
consequence, may result in great economic loss due to reduced efficacy of animal husbandry
(Bata and Lasztity, 1999). Aflatoxins have also been reported to be a potent carcinogen in
rats and often linked with hepatocellular carcinoma (HCC) in many species of animals
(Wogan, 1992). They are acutely toxic, immunosppressive, mutagenic, teratogenic with
severity of effects varying according to dosage (Garuba et al., 2011).
Owing to the various health problems and high economic losses resulting from
aflatoxin contamination of feed and food commodities, there is a need to characterize the
aflatoxigenic fungi associated with various feed and food commodities.
One of the major food and feed commodity in Ibadan is maize (Zea mays) which
serves as serves as a raw material on which many agro-based industries depend on (Garuba
et al., 2011). The FAO in 2005 reported that a total of 5.5million tons of maize was
produced in Nigeria in 2004 (FAO, 2005). Due to the high consumption of maize and maize
products in Nigeria, there is a need to screen the maize grains for aflatoxin contamination as
well as characterize fungi present in this maize samples. This will go a long way in
providing information necessary for the control of aflatoxin contamination in the various
food and feed samples. Therefore, this study was conducted to investigate the distribution of
aflatoxigenic fungi and aflatoxin contamination of maize samples in Ibadan.

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MATERIALS AND METHODS
SAMPLING SITES AND SAMPLE COLLECTION
Twelve maize samples were collected from four agro-ecological locations: Ido Local
Government; Ona-ara Local Government; Akinyele Local Government and Lagelu Local
Government all within Ibadan, Oyo State, Nigeria. Ibadan is the largest city in the Sub-
Saharan African and has tropic vegetation. The climate is characterized by dry November to
April and wet May to October season. Only maize kernels that had been in storage for up to
6 months were collected from the farmers. The maize grains were kept in clean polyethylene
bags and transported to The University of Ibadan Microbiology Laboratory for further
analysis. To prevent further postharvest accumulation of moulds and aflatoxins prior to
analysis, all the samples were stored at 4 °C.
DETERMINATION OF MOISTURE CONTENT
Moisture content of the samples was determined using moisture content analyzer (Sinar
Model 6095 Agri ProTM).
ISOLATION AND IDENTIFICATION AFLATOXIGENIC FUNGI
A portion of each sample was ground using a high-speed blender (Waring Commercial,
Springfield, MO) for 1 min. One gram of sample was weighed, mixed in 10 ml of sterile
distilled water and shook for about 10 minutes. 200 µl aliquot of this was transferred onto
the surface of sterile Potato Dextrose Agar (PDA) plates and incubated at 31 oC for 3 days.
The number of fungal growth exhibiting morphologies consistent with Aspergillus,
Fusarium and Penicillium, species (Singh et al., 1991) were counted. After incubation,
isolates were tentatively classified into species and strains by observing cultural
characteristics and conidial morphology. Observed characteristic features were then
compared with standard text (Cotty, 1989; Klich and Pitt, 1988).
DETERMINATION OF AFLATOXIN PRESENT IN THE MAIZE SAMPLES
A 20-g sub-sample from a bulk sample was ground and extracted with 100 ml of 70%
methanol using a high-speed blender (Waring Commercial, Springfield, MO) for 3 min. The
mixture was then passed through Whatman filter paper No. 1, and the extract collected in a
250 ml separatory funnel and 100 ml of distilled water was added to ease separation. The
solution was extracted twice with 25 ml methylene chloride (dichloromethane). Following
separation, the methylene chloride layer was filtered through 40 g of anhydrous sodium
sulphate to remove residual water. The extract was collected in a polypropylene cup and
evaporated to dryness in a fume hood. The residue was re-dissolved in 200 µl of methylene
chloride and either diluted or concentrated to allow accurate densitometry.
Extracts and aflatoxin standards were spotted on thin-layer chromatography (TLC)
plates (silica gel 60, 250 µm) by development with diethyl ether-methanol-water (96:3:1),
visualized under ultraviolet light, and scored visually for presence or absence of aflatoxin
with a 2 ng limit of detection. Aflatoxins were quantified using scanning densitometer,

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CAMAG TLC Scanner 3 with win- CATS 1.4.2 software (Camag A G, Muttenz,
Switzerland) (Suhagia et al., 2006).
DATA ANALYSIS
Experiments were done in triplicates and data obtained were analyzed using SPSS Version
15.
RESULTS AND DISCUSSION
Table 1 shows the percentage moisture content of each of the maize sample collected for the three local
governments. Sample A from Ido Local Govt had the highest moisture content of 10.2%, closely followed by
samples I and J both from Akinyele local Government with moisture content of 9.3% each. Sample K from
Ona-ara Local Government had the least moisture content of 6.4% (Table 1).
Local Government
Samples
Moisture content (%)
Ido
A
10.2
B
9.2
C
7.5
Lagelu
D
7.0
E
8.2
F
8.8
Akinyele
G
9.3
H
8.0
I
9.3
Ona-ara
J
7.7
K
6.4
L
8.2
Table 1; moisture content of the twelve maize samples collected from four local governments within Ibadan,
Oyo state, Nigeria. Data are means of three replicates. Values followed by the same letters are not significantly
different by Duncan’s multiple range test (P < 0.01).
One hundred and twenty fungi belonging to three different genera and eleven different
species were isolated form the twelve maize samples. Forty-five of the one hundred and
twenty fungi isolated were identified as Aspergillus sp. (45), sixty-one (61) as belonging to
the genus Fusarium and fourteen as species of Penicillum (Table 2) based on the observed
characteristics in comparison with standard text (Cotty, 1989; Klich and Pitt, 1988).
The percentage incidence of the various species of moulds isolated is represented in
Table 3. Aspergillus flavus has the highest percentage occurrence of 20.8%, followed by
Fusarium oxysporum 15%, Fusarium solani 10% which is closely followed by another
species of Fusarium (9.16%) while Penicillum fellutanum had the least percentage
occurrence (2.5%).

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The results of the aflatoxin contamination of the twelve maize samples is presented in Table
4. The results revealed that Fifty eight percent (58%), corresponding to seven out of the
twelve maize samples is contaminated aflatoxin B1. Sample I from Akinyele has the highest
aflatoixn B1 content (24.10) followed by Sample H (19.47) also from Akinyele. Sample H is
closely followed by sample A (18.14) from Ido Local government. Fifty percent (50%), six
out of total twelve maize samples is contaminated with aflatoixn B2 as shown in table 4 with
sample I (Akinyele) having the highest aflatoxin B concentration of 7.67 followed by 4.36
(sample F) and 1.46 from sample B. With respect to Aflatoxin G1 contamination only two
(16.66%) of the twelve maize samples is contaminated with aflatoxin G1 while aflatoxin G2
was detected in only one out of the twelve.
All the twelve maize samples investigated in this study has moisture content that falls
within the within the recommended 12% for storage (Christensen and Kaufmann, 1974).
This moisture content of the maize samples observed in this study is relatively lower than
observed in literatures (Hell et al., 2000; Garuba et al., 2011). This could be an indication of
adherence to good pre- and post-harvest agricultural practices aiming at drying maize grains
rapidly so as to prevent fungal contamination. This reduced moisture content is of great
significance since high moisture content and rich nutritional components of maize has been
reported to encourage mould growth on maize during storage (Garuba et al., 2011).
Despite the reduced moisture content, there is still the contamination of the maize
samples by toxigenic fungi and other fungi as evident by the results. All the toxigenic fungi
isolated in this study have also been previously reported to be associated with stored maize
in Nigeria (Udo et al., 2000; Bankole and Mabekoje 2003, Atehnkeng et al., 2008; Garuba et
al., 2011).
Sample
Aspergillus sp
Fusarium sp.
Penicillum sp.
A
2
17
0
B
2
0
0
C
6
18
0
D
1
3
0
E
2
2
0
F
2
0
2
G
13
2
0
H
6
13
0
I
4
0
2
J
2
5
2
K
2
1
1
L
3
0
7
Total
45
61
14
Table 2; Incidence of mould contamination of the twelve maize samples. Data are means of three replicates.
Values followed by the same letters are not significantly different by Duncan’s multiple range test (P < 0.01).

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Isolates
No. of occurrence
Percentage
Aspergillus flavus
25
20.8
Aspergillus niger
6
5.00
Aspergillus fumigatus
10
8.33
Aspergillus parasiticus
4
3.33
Fusarium sp.
11
9.16
Fusarium dimerum
10
8.33
Fusarium ciliatum
10
8.33
Fusarium oxysporum.
18
15.0
Fusarium solani
12
10.0
Penicillum Jathinellum
4
3.33
Penicillum fellutanum
3
2.50
Penicillum digitatum
7
5.83
Table 3; Percentage occurrence of the different mould species in the twelve maize samples from the four
different local governments within Ibadan, Oyo state, Nigeria. Data are means of three replicates.
Sample source
Sample code
Aflatoxin B1
Aflatoxin B2
Aflatoxin G1
Aflatoxin G2
Ido
A
18.14
0.00
0.00
0.00
B
1.82
1.46
4.89
0.00
C
0.00
0.00
0.00
0.00
Lagelu
D
0.00
0.00
0.00
0.00
E
5.42
1.57
0.00
0.00
F
2.20
4.36
0.00
0.00
Akinyele
G
0.00
0.00
0.00
0.00
H
19.47
1.64
0.00
0.00
I
24.10
7.67
0.00
0.00
Ona-ara
J
4.84
3.30
0.85
10.78
K
0.00
0.00
0.00
0.00
L
0.00
0.00
0.00
0.00
Table 4: aflatoxin content (pbb) of the maize samples from four local governments with Ibadan. Data are
means of three replicates. Values followed by the same letters are not significantly different by Duncan’s
multiple range test (P < 0.01)
The high incidence of Fusarium species in this work could either be as a result of infection
of the maize samples prior harvesting as the case of Fusarium species (which are known to
be fungal pathogens) while the high incidence of Aspergillus and Penicillum sp.
contamination could be due to cross contamination as suggested by Bankole and Mabekoje,
(2003). Also Donner et al. (2006) reported that Aspergillus flavus is the most dominant in
West African soils. The high frequencies of Aspergillus flavus in the soil that acts as the
reservoir may be responsible for the infection of maize grains while in the field (Nesci and
Etcheverry, 2002).
Aflatoxin contamination studies showed that about 58% of the total maize sample is
contaminated with at least one type of aflatoxin investigated in this study with about 31.2 %
of the 58% having aflatoxin contamination above the 5ppb detection limit. Studies of
aflatoxin contamination in Nigerian foods reported contamination higher than data presented
in this report (Setamou et al., 1997; Udo et al., 2000; Bankole and Mabekoje, 2003;
Atehnkeng et al., 2008). It is likely that household maize represent a significant source of
exposure to aflatoxin in Nigeria. The low level of aflatxoin contamination reported in this

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study despite the high incidence of aflatoxin-producing organisms could be a function of the
substrate as substrate contamination by aflatoxin has been reported to be influenced by
factors such as substrate type (Bankole and Mabekoje, 2003). Furthermore, the low level of
aflatoxins could be due to the fungal interactions between the aflatoxigenic fungi and other
toxigenic fungi present within the maize samples. The lack of good storage facilities and
also poor post-harvest management practices induces fungal contamination and
accumulation during post-harvest periods.
CONCLUSION
In conclusion, the results of this work showed the relatively low level of aflatoxin
contamination in some samples of maize grain to be marketed in Ibadan Metropolis. These
results coupled with that of the moisture content of the maize samples are indicative of slight
adherence to the pre- and post-harvest strategies aimed at reducing fungal contamination and
accumulation of mycotoxins in stored agricultural produce. However, because of the
relatively high incidence of other toxigenic fungi such as the various species of Fusarium
and Penicillium there is a need for further screening for the contamination of these stored
maize grains for contamination by toxins such as Fumonisin, trichothecenes and citirinin that
are produced by these organisms.
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ACKNOWLEDGMENTS
The authors are grateful to Mr Alao Omoniyi of the Department of Microbiology, University of
Ibadan for Technical assistance provided during the course of this work.
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