Journal of Microbiology and Biotechnology Research
Scholars Research Library
J. Microbiol. Biotech. Res., 2013, 3 (1):8-13
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ISSN : 2231 –3168
CODEN (USA) : JMBRB4
Isolation of aflatoxin producing species of Aspergillus from foodstuffs sold in
calabar markets, Cross River State, Nigeria
*1 Clement Abriba, 1J. A. Lennox, 1Bassey E. Asikong,1atim Asitok 1 Ikpoh S. Ikpoh,
1
Effiom E. Henshaw, 2matthew E. Eja.
1
2
Department of Microbiology, University of Calabar, Calabar, Nigeria
Department of Biological Science, Cross River University of Technology Calabar,
Nigeria.
______________________________________________________________________________
ABSRTACT
The isolation of aflatoxin producing species of Aspergillus from foodstuffs sold in calabar market was investigated.
A total of eight food samples were bought from markets within Calabar. The samples were separately analysed for
the presence of aflatoxin producing species of Aspergillus. The isolation of these species was carried out using
dilution plating and direct plating methods respectively. Altogether thirteen species of moulds belonging to three
genera were isolated from these samples. The most predominant genus-Aspergillus accounted for ten species, while
Penicillium accounted for two species and Rhizopus accounted for one species. The aflatoxin producing species of
Aspergillus, A. flavus was isolated from six food samples (peanuts, rice, groundnuts, wheat, maize and flour) while
A. parasiticus was isolated from four food samples (peanuts, rice maize and groundnuts). However, there were
variations in the degree of occurrence of each of the two species in each of these samples.
Key Words: Aflatoxin, Aspergillus species, food stuffs.
______________________________________________________________________________
INTRODUCTION
Aspergillus is a saprophytic mould (Madunagu and Umana 2000). It is known as a filamentous fungus (Disalvo
2000; Woods 2002). It was originally classified as Deuteromycotina (Fungi imperfecti) but further research
reassigned it to Ascomycotina (sac fungus) because of its ability to form asci within closed ascocarps (Porter, 2001).
However, the genus Aspergillus has not been subdivided into classes or orders because of the fact that
Ascomycotina classification was not complete. But today the genus Aspergillus has been given complete
classification (Dixon and Fromtting, 1991).
Morphologically, Aspergillus fungi are monomorphic but display a complex regulated development. They have
septate hyphae and conidia. They form fruiting bodies that are spherical or flask or cup shaped and whose sexually
formed spores are borne in sacs called asci. They are the largest group of fungi that produce both sexual and asexual
spores and conidia (Balbach and Bliss, 1982).
There are over 200 different species of Aspergillus. Some of the more common ones include: Aspergillus niger, A.
flavus, A. carbonaceous, A. terreus, A. parasiticus and A. tamari. Aspergillus is found in various habitats under
different environmental conditions. Some strains of Aspergillus are used in producing antibiotics and some
beneficial genetic manipulation mechanisms.
They are also used in food fermentations, production of citric acid, used as preservation in soft drinks and canned
foods, also as sources of enzymes (Balbach and Bliss 1982, Porter, 2001).
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Clement Abriba et al
J. Microbiol. Biotech. Res., 2013, 3 (1):8-13
______________________________________________________________________________
Inspite of these merits, there are some demerits associated with this fungus. Aspergillus tends to cause the spoilage
of foodstuffs and can decompose other materials such as woods, textiles (Gupeta, 1981) paints and while some
species severely damage agricultural crops by producing secondary metabolites called aflatoxin.
Aflatoxins are toxic by-products which are group of related difuranocoumarin, produced by the toxigenic strains of
Aspergillus flavus, and A. parasiticus during their growth on crops such as corn, peanuts, wheat, vegetables and
other tree nuts (U. S. department of Agriculture 1990, Cary et. al., 1998). It is the most hepatotoxic and carcinogenic
toxin of fungal origin (U. S. department of Agriculture 1990). It is the most widely and intensively studied group of
mycotoxins and the second most abundant fungal toxin (U. S. department of Agriculture 1990)
There are about ten types of aflatoxins known. Aflatoxin B (AFB) is the most abundant and toxic member of the
group. AFBI is found to be toxic to all animals tested and it is the most hepatocarcinogen known (Squire 1981). The
toxicology of aflatoxin has been well documented for several animal species and humans. The signs of aflatoxin are
diverse and numerous and often depend on species, sex, age, stress, reproduction and health status of the animal and
humans. They include cancer, reduced immune function with compromised resistance to infections and diseases
(Charmley and Trenholm 2000).
Aflatoxin producing strains of Aspergillus usually contaminate crops both in the field and when they have been
harvested and stored (Hudler 1998; Far et al., 1989; Norton 1995).
In general, it appears that in those areas of the world where humidity is high or moderate and temperature are
moderately high and where harvesting, storage and marketing facilities are primitive, there is considerable frequency
of contamination of foods and feeds by aflatoxin producing species of Aspergillus (Hunter 1989; Charmley and
Prelusky 1995).
Calabar is an area that experiences rain almost all through the year. The temperatures are also high. These conditions
favour the growth of Aspergillus species in stored grains, foodstuffs feeds, which are later, sold to the public.
In this study we sought to isolate and identify aflatoxin producing species of Aspergillus from various foods sold in
Calabar markets to consumers.
The significance of this study therefore lies in the fact that knowing those foods that are contaminated with aflatoxin
producing species of Aspergillus, it will then be possible to offer suggestions to local foodstuffs sellers and
consumers on how to handle and or prevent contamination of the foodstuffs, so as to avoid out break of food
intoxication.
MATERIALS AND METHODS
Sample Collection
Eight different types of food samples (Peanuts rice, Maize/ corn, groundnuts, flour, scorn wheat and milk) were
bought from local sellers in Marian and Watt markets, all in Calabar, Nigeria. The samples were asceptically
collected in sterile polytene bags and taken to the laboratory where they were kept in a refrigerator maintained at
40C for not more than three days before the analysis.
Media
The following media were used: Saboraud Dextrose Agar (Oxoid, Difco, USA) (SDA), potato Dextrose salt agar
(Oxoid, Difco, USA) and Aspergillus differential medium (Oxoid, Difco, USA).
They were sterilized at 1210C for 15 minutes in an autoclave. The media were cooled to 450C and 1ml from 100ml
of streptomycin solution was added to the media to obtain 40ppm concentration of the antibiotic in the media. This
was done to increase selectivity by inhibiting bacterial growth.
Isolation of Aflatoxin producing Aspergillus
The methods used in the isolation of aflatoxin producing species of Aspergillus were:
(i) The dilution method of Warcup (1960), as modified
(ii) Direct plating method of Mislivec and Stack (1984). Here we employed two techniques for the analysis of
foodstuffs.
(a) Analysis of non-surface disinfected (NSD) foods
(b) Analysis of surface disinfected foods
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J. Microbiol. Biotech. Res., 2013, 3 (1):8-13
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RESULTS AND DISCUSSION
TABLE 1:Characterisation of the fungal isolates from the eight food sampes analysed
COLOUR OF COLONY
Bright green with
Black sclerotia
Bright green
Tan to brown
NATURE OF HYPHAE
Septate
Septate
Septate
Septate
NATURE OF CONIDIOPHORE
Hyaline thick walled and
Coarsely roughened
Coarsely roughened
Flexous, smooth, walled and colorless
Blue green
Black
Septate
Septate
Dark Green
Septate
Smooth, walled greenish in the upper portion
Smooth, walled, colourless
With browinish shade on upper half
Smooth and colourless
NATURE OF CONIDIA
Globose and spiny
Smooth and pigmented
Rough and pigmented
Globose to slightly
Elliptical
Globose to subglobose
Globose, thick walled
Brown irregularly rough
Slight roughen
COLOUR OF SUBSTRATE HYPPHE
Orange
PROBABLE ORGANISM
Orange –red
Purple
Aspergillus flavus
A. Parasiicus
A. terreus
Blue to black
Pale Yellow
A. fumigatus
A. niger
Purple
A. vesicolor
TABLE 1 CONTD
Bright Yellow with
Purples sclerotia
Deep chocolate brown
Septate
Spiny and Yellow Pigment
Septate
Dark green
Yellow green
Blue –gray
Dark, green black
Dar –gray
Septate
Septate
Septate
Septate
Asepate
Hyaline, thick walled
Coarsely roughened
Smooth walled and brown Pigmented
Smooth walled and brown Pigemented
Biverticiliate
Assymetrical
Wooly grayish Smooth walled
Smooth walled, Globose
to slight elliptica
Double wall and Colour bars
Purple
A. ochraceus
Pinkish
A. tamarii
Globose and rugolose
Smooth and elliptical
Globose and smooth
Elliptical and Smooth
Globose black in colour
Purpish-red
Yellow
Yellow
Yellow-green
Gray
A. nidulans
A. oryzae
Penicillium citrinum
P. oxalicum
Rhizopus spp
TABLE 2: Molds isolated from each sample analysed using dilution plating method and direct plating method
MOULDS ISOLATES
Aspergillus flavus
A. parasiticus
A. terreus
A. funigatus
A. niger
A. vesticolor
A. ochraceus
A. tamari
A. nidulans
A. oryzae
Penicillum spp
Rhizopus spp
PEANUT
+
+
+
+
-
RICE
+
+
+
+
+
+
+
-
MAIZE
+
+
+
+
+
+
+
+
GROUNNUT WHEAT
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
= ISOLATED; -
SCORN FLOUR
+
+
+
+
+
+
+
+
+
+
=NOT ISOLATED
MILK
+
+
-
TOTAL NUMBER OF FOOD SAMPLES
6
4
3
3
7
2
4
3
2
3
4
3
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Clement Abriba et al
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______________________________________________________________________________
Samples for analysis of non-surface disinfected food technique were kept in refrigeration at 200C for 72 hours before
plating. This destroys mites and ants which serve as agent of spread of mould and thus ensures accurate results from
the analysis.
Identification of Aflatoxin producing isolate
The identification of aflatoxin producing species of Aspergillus was based on macroscopic and microscopic analysis.
The macroscopic identification involves observing the cultural characteristics of each species in the plates, such as
colour of colonies. Aspergillus flavus is clearly differentiated from A. parasiticus and other Aspergilli by producing
a characteristic orange pigment in Aspergillus differential medium (Humsa and Ayres 1977; Pitt et al., 1983;
Hocking and Pitt 1986 and Klich and Pitt 1988).
The microscopic analysis involves the direct identification of the Aspergillus species on the medium by deligent
manipulation with low power (Mislevic, 2000); Preparation of wet mount from the culture plate using lactophenol
blue and covered with coverslip and then examined using low power. In this microscopic examination, the
distinctive features of each species are clearly shown namely, the nature of the hyphae, nature of conidiophore and
the nature of the conidia. The identification was further confirmed by determining the percent G+C of the isolates.
The results from this present study are shown on table 1 and 2. The investigation has shown that the most
predorminant genus Aspergillus accounted for 10 species. This explains why it is called ubiquitous moulds which
are capable of growing in a wide variety of foods, tree crops and fabric (Chamley and Prelusky 1995, Cheesbrough
2000), while Pencilluim had 2 species and Rhizopus one species. Aspergillus, Penicilluim and Rhizopus accounted
for 76.92%, 15.39% and 7.69% respectively of the species isolated. Aspergillus, the genus with the highest number
of isolates occurred in 99% of the samples analysed while Penicillium had 50% and Rhizopu 37.5% respectively of
occurrence of the total samples analysed.
The aflatoxin producing species of Aspergillus. A.flavus was isolated from six food samples (Peanuts, rice,
groundnuts, wheat maize/corn, and flour), and A. parasiticus from four samples (peanuts, rice, maize/corn and
flour), Table 2. The absence of A. parasiticus in wheat and flour is in contrast to the findings contained in U. S.
department of Agriculture (1990), which stated that for aflatoxin to be produced, that A. parasiticus and A. flavus
must be found growing together. A flavus was clearly differentiated from A. parasiticus and other aspergilli by the
production of a characteristic organge pigment in Aspergillus differential medium. It was further confirmed by
determination of percentage G+C of the isolate. This observation is in line with the reports of Hocking and Pitt
(1989) and Klich and Pitt (1988), Pitt et al 1983, and Charmley and Prelusky 1994).
Culturally, Aspergillus species require 1-3 weeks for growth. The colonies begin as a white dense mycelium, which
later assume a variety of colours according to the species based on the colour of their conidia. This observation is in
consonance with the findings of Disalvo (2000). The species isolated were monomorphic but they displayed
complex regulated developments (Woods 2002).
More colonies of the aflatoxin producing species of Aspergillus were obtained from maize/corn, groundnuts and
flour. This is because the samples were suitable for the growth of the species since they contain optimum pH and
optimum moisture content (MC) as stated by Adebayo et al (1994). Also, their presence in the maize/corn and
groundnuts were enhanced by the aerobic condition of the substrate as reported by Charmley and Prelusky (1995)
and Mislivec (2000).
Some of the conidia of Aspergillus species isolated were either hyaline or pigmented, smooth or rough with single or
double wall (Table 1). This observation is in harmony with the work of Mislivec. (2000)
From the dilution plating method employed, it was observed that many colonies were obtained by spread plate
technique than the pour plate one. This is because the colonial morphology of surface colonies is easily observed,
also organisms are not exposed to the heat of the melted agar medium so higher counts are obtained in spread plate
technique as stated by ICMF, 1978 and Mislivec and Stack (1984).
The application of direct plating method for the samples (wheat, rice, peanuts, groundnuts and maize/corn) which
was held with forceps suggested that the contamination of these samples was both external and internal invasion.
This result supports the use of direct plating method for comparison of contamination of food sample as described
by Mislivec and Stack (1984). For instance, A. flavus and A. parasitius showed a high incidence in both non surface
disinfected (NSD) and surface disinfected (SD) foods analysed.
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______________________________________________________________________________
No bacterial contamination was observed. This was as a result of the addition of antibiotic solution to the media as
described by Mislivec (2000) and also as a result of the addition of salt and processing at 200C for 72 hours to
prevent mould seeds (Mislivec and Stack 1984).
Finally, the higher number of colonies obtained from maize/corn and flour samples may also be linked to late
harvesting, insect damage, improper storage conditions improper processing, use of contaminated equipment, poor
transportation facilities and unhygienic personnel handling the milling process. It is therefore necessary to process
these foodstuffs properly, after purchase, in order to avoid food intoxication that could result from the consumption
of improperly processed foodstuffs.
CONCLUSION
The presence of aflatoxin-producing species of Aspergillus and other non aflatoxin producing species in foods and
foodstuffs should be of great concern to the producers, sellers and consumers because they can cause deterioration
and decomposition which can result in substantial economic loses and also could pose potential health hazards to
humans and animals because of their ability to produce toxic secondary metabolites known as aflatoxins which are
carcinogens amongst other toxins. Further work could therefore be carried out to determine the level of these toxins
in the foodstuffs.
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