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SCREENING FOR THE PRESENCE OF Aspergillus flavus AND Aspergillus

parasiticus IN MAIZE GRAINS SOLD IN FIVE SELECTED MARKETS IN


DAR ES SALAAM.

ADOLF Ismael C
2011-04-04819

Submitted in fulfillment of the requirements for the Degree award of Bachelor of


Science in Molecular Biology and Biotechnology
Department of Molecular Biology and Biotechnology
College of Natural and Applied Sciences,
University of Dar es Salaam
June, 2014.

SUPERVISORS
ACADEMIC SUPERVISOR:

Dr. MUGASSA S.T.


RUBINDAMAYUGI

RESEARCH &TECHNICAL SUPERVISOR:

Mr. CHUWA

ABSTRACT
Maize consumption is one of the primary avenues through which humans become exposed to
aflatoxin contamination especially in developing countries. This study was conducted to screen
for the presence of potential Aflatoxigenic fungi in the five selected markets in Dar es Salaam.
The medium, Aspergillus Flavus and Parasiticus Agar (AFPA) medium was used as differential
medium to screen for A. flavus and A. parasiticus. All collected samples tested positively on
AFPA medium. However, the comparison of level of contamination among markets as suggested
in specific objective (ii) was not possible due to contamination of colonies with maggots on
AFPA medium.
Key words: Aspergillus flavus, Aspergillus parasiticus, AFPA, aflatoxin, maize, mycotoxins

1.0. INTRODUCTION
1.1. General Background Information
Aspergillus is a hyphomycetous genus of fungi that have many species inhabiting various
niches. Some of the common species includes, A. flavus, A. parasiticus, A. japonicas, A.
fumigates, and A. niger. Mainly they occur as saprophytes in soils, stored food and feeds.
Aspergillus species contaminate different food commodities including maize grains, groundnuts,
peanuts, rice, wheat, cassava and a number of various fruits. Aspergillus can contaminate
agricultural products before and after harvest. They prefer growing in humid warm tropical
climates, a common climate found in most parts of Africa.
Countries such as Tanzania that is located between 400 N and 400S latitude offer suitable growing
conditions for the fungi. Since Aspergillus spp. originates in the soil, the risk of contamination

begins with planting, and can be worsened later through inappropriate harvesting, handling,
storage, processing, and transport practices. Contamination during crop development and after
harvest depends on environmental conditions that are optimal for the growth of fungi. Damage
by pests (birds, mammals, and insects) or the stress of hot, dry conditions can contribute to
significant Aspergillus infection. Drought stressors (elevated temperature and low relative
humidity) increase the number of Aspergillus spores in the air, increasing the chance of
contamination. Heavy rain can cause spores to splash onto fruit and grain. After harvest, high
crop moisture coupled with warm temperatures, inadequate drying, and poor storage can further
increase the risk of contamination (Tanzania food and Drug authority, 2012).
Although many Aspergillus species are saprophytic, some are parasites on plants, animals and
insects. Aspergilli are rarely directly pathogenic to plants in fields (Mohammadi et al, 2008).
Aspergilli have both positive and negative impacts on the economy. The most notable positive
economic effect of Aspergilli is the production of useful enzymes that are utilized in food
industries Amylase and citric acid are two of the most important products produced by some
Aspergillus species. However, some species produce toxic secondary metabolites that incur
negative impact on economy. These metabolites are called mycotoxins and they are potent
harmful toxins on human and animal health.
There are various kinds of mycotoxins produced by various genera and species of fungi. The
mycotoxins include aflatoxin, Ochratoxin, Zearalenone, Trichothecenes, Patulin and Fumonisins.
The important genera considered to produce mycotoxins in foods are Aspergillus, Penicillium,
Fusarium, Alternaria and Cleviceps. (Sekar et al, 2008).

However, Aflatoxin is the most economically important mycotoxin throughout the world, it is
mostly produced by A. flavus and A. parasiticus. The most toxic form of aflatoxin is aflatoxin
B1. Other forms of aflatoxins are aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1 and
aflatoxin M2. These forms differs from each other basing on their chemistry which give different
fluoresces characteristics under UV light. Aflatoxin B fluoresces blue while aflatoxin G
fluoresces green. Aflatoxin M is derived from from aflatoxin B when consumed by animals and
it fluoresces blue as well. It is commonly found in milk and blood of cattle and it is potentially
harmful. Aflatoxins have hepatotoxic, carcinogenic,and teratogenic potential that can affect
either humans or animals.
Aflatoxin production by aflatoxigenic Aspergillus species is influenced by different factors.
Some studies have been conducted to investigate conditions favoring aflatoxin production in
vitro. The work published by Ritter et al, 2011 showed that different Aspergillus flavus strains
responded differently on different culture condition on the production of aflatoxin. The
conditions that were manipulated were nutrients, temperature, pH and time for incubation.
However, not all strains of Aspergillus flavus produce aflatoxins. Some strains do not produce
aflatoxins at all regardless to the environmental factors.

1.2. Statement of the Research Problem


Maize is the most widely grown crop in Tanzania, representing 82% of all Tanzanian farmers
(National Sample Census of Agriculture, 2002-2003). 41% of Tanzanians calories are contributed
by maize consumption (Abt, 2012). The combination of high production and high consumption
of the maize raises a great safety concern when the maize grains are contaminated with aflatoxin
producing Aspergilli. This implies that even at low level of aflatoxin in the maize grain or

product can cause health effect because of daily and prolonged exposure. Therefore, there is a
high need of assessing the maize for aflatoxin or aflatoxin-producing Aspergillus species
contamination.
There have been a number of reports on liver diseases and different forms of carcinogenesis in
Tanzania with unknown exactly causes. In 2010, WHO reported 1209 liver cancer cases in
Tanzania. Although these problems can be caused by different agents like exposure to strong
radiations, carcinogenic chemicals discharged in the environments and viral infection, there is a
high incidence to be caused by consuming aflatoxin contaminated crops, meat and milk because
aflatoxin is carcinogenic and is associated mainly with hepatocarcinogenesis.

1.3. Objectives
1.3.1. General Objective
To Screen for the presence of Aspergillus flavus and Aspergillus parasiticus in maize grains sold
in five selected markets in Dar es Salaam
1.3.2. Specific objectives
i) To isolate A. flavus or A. parasiticus from maize grains sold in five selected markets.
ii) To compare the contamination level of Aspergillus species between the selected markets.

1.4. Significance of the Study


This study will generate general awareness about Aspergillus flavus and Aspergillus parasiticus
contamination which subsequently indicates the likelihood of contamination with aflatoxins. The
study will show the status of maize crops in the markets basing on Aspergilli contamination view

point, which can reflect the status of contamination in the production environments where a large
proportion of maize is consumed by producers (farmers). Understanding about aflatoxigenic
Aspergilli contamination and associated effects, farmers and local sellers can adopt a proper
management of the post- harvest and utilization that will minimize the contamination of
commodities with Aspergillus species. Overall, as people become aware of all these and
convinced to adopt the practices that minimize the Aspergilli contamination, the problems
associated with mycotoxicoses can be significantly reduced.

1.5. Research Questions


i) Are the maize grains in the markets contaminated with A. parasiticus and A. flavus?
ii) Is the level of contamination among markets similar?

2.0. METHODOLOGY
2.1. Study Site
The study was conducted in Dar es Salaam. The maize grains samples were collected from five
local markets in Dar es Salaam. The markets were Tandale, Kariakoo, Buguruni, Temeke and
Mbagala.

2.2. Research Protocol


2.2.1. Sample collection and Preparation
10 g of maize grains were collected from 10 different sellers in the same market stores randomly.
These samples were mixed to give 100 g of the total sample and transported in the nylon
container. In the laboratory, the sample was divided into five sub-samples, each about 20 g and

stored at temperature of 180 C. The same procedure was used for other samples from the
remaining market.
Each subsample was taken in the conical flask and washed with 3% Sodium hypochlorite
(NaOCl) for 5 minutes for surface sterilization and subsequently washed in distilled water, and
allowed to dry for 45 min prior to plating.
2.2.2. Media Preparation
Two solid media were prepared. These are Malt Extract Agar (MEA) medium and Aspergillus
Flavus and Parasiticus Agar (AFPA) medium. To prepare, MEA medium, 3 g of commercial malt
extract agar were dissolved into 300 ml of distilled water. The contents were boiled to mix
thoroughly and then autoclaved at 1210C and 1 atm.
To prepare AFPA medium, 3 g of peptone, 6 g yeast extract, 0.15 g Ammonium ferric citrate, 4.5
g agar and 0.01 g amphicilin were dissolved into 300 ml water. The contents were mixed
thoroughly though boiling and then autoclaved at 1210C and 1 atm.
2.2.3. Fungi Isolation
6 surface sterilized maize grains from each sub-sample were plated onto a petri-dish containing
Malt Extract Agar (MEA) medium. The plates were incubated in the incubator at 300 C for 5
days. The kernels showing the growths of fungi were identified, and each colony on each kernal
from each plate was sub-cultured on differential media; AFPA medium for isolation of
Aspergillus flavus and Aspergillus parasiticus. The plates were incubated at 300C for 5 days. The
yellow and orange colonies which represent the colonies derived from A. flavus and A.
parasiticus were identified.

2.2.4. Microscopic Observation


A sample of colonies from AFPA medium was wet smeared onto a slide and observed under light
microscope at 100x. The photograph for appearance of fungi was taken.
3.0. RESULTS
3.1. Colony appearance

Fig.1. Appearance of fungi colonies on maize kernals grown on MEA medium on some
plates.

Fig.2. Reverse appearance of colonies on AFPA medium of selected plates.

Fig.3. Appearance of isolated Aspergillus spp under light microscope

4. DISCUSSION
The samples from all markets were screened for the presence of A. flavus and A. parasiticus by
AFPA medium. AFPA contains Ferric ions which reacts with Aspergillic acid produced by both
A.flavus and A.parasiticus forming yellow or orange pigments on agar medium (Assante et
al.,1981). From data collected, all samples from all markets show AFPA positive results. This
implies that these species, either A.parasiticus or A. flavus or both are wide spread in maize
grains sold in markets. This also reveals the status of the maize in the production areas who are
the main consumers of the maize because the inoculation normally starts in the field.
Aspergillus flavus forms sclerotia which allow it to survive saprophytically in the soil, maize
residues and cobs for extended periods of time (Schedegger and Payne, 2003).
From the results, maize shows to be commonly contaminated by Aspergillus flavus or /and
Aspergillus parasiticus. This is in agreement with a study conducted by Muthomi et al.,2009 and
Gao et al.,2007 which show that Aspergillus flavus are the common contaminating fungi of
maize grains and maize products. This poses a high risk to the public because maize
consumption is very high and these Aspergilli are potentially producer of aflatoxin. For example,
in many boarding schools, the main food consumed by pupils almost every day is stuff porridge
(Ugali), which is a product of maize. Thus even if the maize have got a low level of aflatoxins,
the daily exposure can lead to long term chronic effects.
The occurrence of A. flavus or A.parasiticus in these markets maize could have been due to
relatively warm condition that dominates almost throughout the year. Normally, the conditions
favoring Aspergilli growth does favor aflatoxin production, but fungi growth can occur with little
or no mycotoxin production (Stack and Carlson, 2006). This means that, the presence of
Aspergillus flavus or Aspergillus parasiticus on maize does not imply an obvious occurrence of

aflatoxin in the maize. Thus, there is a high need to test the actual presence of aflatoxins in the
maize grains and other susceptible food materials rather than relying on the presence of these
potential aflatoxins producers.
However, the results show that colonies on the AFPA medium are contaminated with the
maggots. The movement of these maggots in the culture disturbs the normal pattern of subcultured colonies that shows AFPA positive property. Through the movement of maggots in the
culture, the fungi are swept to other locations or points of the medium establishing new colonies.
This phenomenon makes impossible to enumerate the number of developed colonies in a given
plate and compare it with the developed colonies in other plates. Because of this problem, the
attainment of specific objective (ii) becomes impossible. Maggots are larval stage of some
insects particularly flies. This problem might be caused by contamination originated when the
maize grains were being dried on a nylon sheet onto a table after treating the grains with 3%
NaOCl in the laboratory. The access of flies on these maize grains being dried can be easy and if
they lay eggs onto the maize the maggots develop later as the culture is incubated. The
occurrence of flies in the microbiology laboratory is evident.
The results of this study show that there is need for continue Aspergilli and aflatoxin awareness
creation among the sellers, consumers and farmers of maize. Farmers should be educated on the
proper cultivation practices that minimize the exposure of maize to the Aspergilli in the farm
such as avoiding the maize contacting the soil while harvesting (Muthomi et al.,2009). The maize
should be dried enough before being packaged to make sure moisture which is needed for
Aspergilli growth is not available. Sodium Calcium Aluminosilicate can be incorporated in maize
especially that meant for cattle feeds because this compound binds the available aflatoxins
making them unavailable in all portion of the maize sample (Agrios, 2005). It is important to

feed the cattle with the feeds free of aflatoxin because these toxins are very stable, they can
persist in the food chain.
5. CONCLUSSION AND RECOMMENDATIONS
The isolated fungi from all collected maize samples tested positively on AFPA medium. This
indicates the presence of either A. flavus or A. parasiticus or both in the maize samples. This
also reveals that a large portion of the maize grains in the markets are contaminated with these
Aspergillic fungi, which is a scare to the public.
There is a need to repeat this kind of study in different seasons in order to identify the season in
which the fungi growth in the maize predominates in the markets. Also, the study should
incorporate the strategies to identify aflatoxins in the maize as not all strains of A. flavus and
A.parasiticus produces aflatoxins.
Furthermore, the studies on development of maize varieties resistant to Aspergilli species should
be emphasized. Biotechnology seems to have a good promise in solving problems associated
with aflatoxins through genetic engineering.

6. ACKNOWLEDGEMENT
I wish to acknowledge University of Dar es Salaam for giving me the opportunity to advance in
my education. I extend my thanks to Molecular Biology and Biotechnology Department staffs
for preparing me to be competent in academic matters relating to life sciences and research.
My sincere thanks go to my supervisors, Dr. Mugassa S.T. Rubindamayugi and Mr. Chuwa for
all their indispensable support they gave to me in all period of my research project.

My appreciation goes to my family members; my father, mother, brother and sisters for all their
love they show to me. My special thanks go to my mother for her moral support.
Lastly Im thankful to all my colleagues, Molecular Biology and Biotechnology students
(2011/2014) for their cooperation and love they showed to me. God bless you all.

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