ABSTRACT
Aflatoxins are natural toxigenic metabolites frequently found in millet and cereal products. Aspergillus species are mainly responsible for Aflatoxin build-up and contamination of millet products. The main objective of this study was to detect aflatoxin-producing moulds in millet grains commonly sold in Ndoru Market, Umuahia, Abia State. Four (4) millet samples were bought from four (4) different sales point in Ndoru market from which eight (8) organisms were isolated using spread plate technique and belong to 2 genera and 4 species; Aspergillus niger, Aspergillus sp., Penicillium sp., Penicillium sp. The fungal population in millet products were enumerated and characterized using cultural and biochemical techniques. The percentage of occurrence is; A. niger was found in (37%), Aspergillus sp. (25%), Penicillium sp. (25%), Penicillium sp. (12.5%). However the highest prevalence of occurrence was seen in A. niger. Aflatoxin production by isolated fungi was subsequently evaluated using the thin layer chromatography technique and viewed under UV light. Two (2) fungal isolates (Aspergillus niger, Aspergillus sp.) of the Aspergillus genera produced green fluorescence as detected in the culture filtrates while the remaining two (2) isolates (Penicillium sp.) of the Penicillium genera produced no fluorescence. The green fluorescent colouration emitted under UV light which indicates the ability to produce aflatoxin, by aflatoxigenic strains was not observed for any of the tested non aflatoxigenic isolates. The findings from this study suggest that millet products obtained from the two(2) out of the four (4) different sales point are contaminated by aflatoxigenic fungi responsible for producing aflatoxin which could be unsafe as food or feed ingredients and it is recommended to use varieties resistant to toxigenic fungi.
TABLE
OF CONTENTS
Title Page i
Certification ii
Dedications iii
Acknowledgements iv
Table of Contents v
Lists of Tables x
Lists of Figures xi
Lists of Plates xii
Abstract xiii
CHAPTER ONE
1.0 INTRODUCTION 1
1.1 Statement
of the Research Problem. 3
1.2 Aims
and objectives of the study 4
1.2.1 Aim of the study 4
1.2.2 The
objectives of this study 4
CHAPTER TWO
2.0 LITERATURE REVIEW 5
2.1 History
of aflatoxin 5
2.2 Aflatoxin 6
2.2.1 Types
of aflatoxins 6
2.3 Aflatoxin producing fungi 6
2.4 Identification
of Aspergillus Species 7
2.4.1 Microscopic features of Aspergillus species 8
2.4.1.1 Aspergillus
flavus 8
2.4.1.2 Aspergillus
parasiticus 8
2.4.1.3 Aspergillus
fumigatus 8
2.4.1.4 Aspergillus
niger 8
2.4.2 Macroscopic
Features of Aspergillus species 10
2.4.2.1 Aspergillus
flavus 10
2.4.2.2 Aspergillus
parasiticus 11
2.4.2.3 Aspergillus
fumigatus
11
2.4.2.4 Aspergillus
niger 11
2.5 Factors
favouring aflatoxigenic fungal growth and aflatoxin production 12
2.5.1 Physical
factors affecting aflatoxin production. 12
2.5.2 Chemical
factors affecting aflatoxin production 13
2.5.3 Biological
factors affecting aflatoxin production 13
2.6 Human
exposure to aflatoxin 14
2.7 Pathogenicity and Clinical Significance
of Aflatoxin in Man and Animals. 14
2.7.1 Acute
aflatoxicosis in human 14
2.7.2 Chronic
aflatoxicosis in humans and animals 15
2.8 Impact
of aflatoxin on the liver 15
2.9 Health
consequences of aflatoxin16
2.10 Incidence
of aflatoxin poisoning through food consumption 17
2.11 Strategies
for prevention and control of aflatoxin contaminations 18
2.11.1 Creating awareness of aflatoxin effects on
humans 18
2.11.2 Prevention through pre-harvest handling 19
2.12 Biological Control of Aflatoxin
Contamination of Crops 19
2.12.1 Competitive displacement by atoxigenic Aspergillus strains 19
2.12.2 Plant extracts as fungal disinfectant 20
2.13 Prevention
of fungal infestation in food through post harvest handling 21
2.13.1 Drying of food samples 21
2.13.2 Smoking of food 21
2.13.3 Physical separation of suspected contaminated
millet or grains and hygiene 21
2.13.4 Good storage strategies 22
2.13.5 Processing 22
2.14 Chemical treatments for aflatoxin
intoxication 23
2.14.1 Fumigation 23
2.14.2 Ammoniation 23
2.14.3 Enterosorption 24
CHAPTER THREE
3.0 MATERIALS
AND METHODS 25
3.1 Sample
collection 25
3.2 Materials
for fungi culture and microscopy 25
3.3 Materials
for TLC 25
3.4 Preparation
of culture media 25
3.4.1 Sabouraud
dextrose agar 25
3.5 Sterilization 26
3.6 culture 26
3.7 Isolation
And Identification 26
3.7.1 Macroscopic examination of culture 27
3.7.2 Microscopic examination of culture 27
3.8 Screening
of aflatoxigenic fungi 27
3.9 Determination
of aflatoxin using Thin Layer Chromatography
(TLC) technique 28
3.9.1 Method For TLC 28
CHAPTER FOUR
4.0 RESULTS 29
CHAPTER 5.
DISCUSSION, CONCLUSION AND RECOMMENDATION 35
5.1
Discussion 35
5.2
Conclusion 38
5.3
Recommendation 39
References
Appendix
LIST
OF TABLES
Tables Title Page
2.1: Microscopic
properties of Aspergillus species 10
2.2: Colonial properties of Aspergillus species 11
4.1; Results
of morphological and microscopic examination of fungi isolated from millet
samples from different sales point in Ndoru Market. 30
4
2; Results
of fungi isolated from millet samples from different sales point 31
4.3; Occurrence
and incidence of fungi isolated from the millet samples. 32
4.4; Identification
of aflatoxin producing fungi by their fluorescencing characteristic. 34
LIST
OF FIGURES
Figure. Title
Page
4.1 Occurrence of Fungal isolates
from the millets. 33
PLATES
Plate. Title Page
Plate 1: Macroscopic appearance of Aspergillus niger on Sabauroud
dextrose agar 45
Plate 2: Macroscopic appearance of Aspergillus
sp 45
Plate 3: Macroscopic appearance of Penicillium
sp 45
Plate 4: Green fluorescence color emited
by filtrate of Aspergillus niger
and
Aspergillus
sp. under UV light 46
Plate 5: Green fluorescence color as shown
in the TLC plate 46
CHAPTER ONE
1.0
INTRODUCTION
Aflatoxin, a word derived from Aspergillus
flavus toxin is naturally occurring
mycotoxins that is produced by Aspergillus flavus and Aspergillus parasiticus species of fungi. Aflatoxin is
a highly toxic secondary metabolite that contaminates a number of crops causing
a great economic loss). Several other moulds and fungi species also produce
aflatoxin and among them include A. nomius, A. pseudotamarii and A. bombycis.
Millet (Pennisetum spp.)
contributes about 20% share of the main staples in Nigerian average food consumption in calories term. It is commonly
consumed as pap, porridge, local cake (“masa”), millet meal(“tuwo”), gruel-like
drink (“kunu – zaki”), and “fura” in the Northern Nigeria where it is mostly cultivated. The susceptibility of millet
to fungal growth and mycotoxin contamination has been demonstrated in many
parts of Nigeria (Okoye, 2014).
Aflatoxins have been found in soil as well as
in grains, nuts, dairy products, tea, spices and cocoa, as well as animal and
fish feeds. Mycotoxins, the secondary
metabolites from toxigenic fungi, are contaminants in foods and feeds, exerting
harmful effects upon animal and human health (Zahoor-ul-Hassan et al.,
2010). Mycotoxins are low-molecular weight secondary metabolites, produced by
certain strains of filamentous fungi, such as Aspergillus , Penicillium and Fusarium,
which invade crops in the field. most important mycotoxins in naturally
contaminated food and feeds are
aflatoxins (AFs), ochratoxins (O), zearalenone (ZEN), T-2 toxin, deoxynivalenol
and fumonisins (Sultana and Hanif, 2009). Being potential contaminants of food
items in Nigeria, it is important
that more focus is given to their accurate identification in order to have a
grasp of the intervention strategies necessary for food safety and security.
Aflatoxins are serious secondary metabolites that have two possible
designations, B or G, which relate to fluorescence under exposure to long-wave
ultraviolet (UV) light. B aflatoxins appear blue in colour, while G aflatoxins
appear green in colour due to differences in their chemical structures. B aflatoxins occur more frequently as
contaminants, and are also believed to be more potent, than G aflatoxins.
Aflatoxins produced by the toxigenic fungi, mainly Aspergillus flavus and Aspergillus parasiticus, constitute one of the major
health hazard groups of naturally-occurring toxicants, both for man and
animals. Human exposure to aflatoxins may result from consumption of
plant-derived foods that are contaminated with the toxins, and the carry-over
of aflatoxins and their metabolites in animal products such as meat and
eggs. Aflatoxin is one of the numerous
naturally-occurring mycotoxins that are found in soils and foods. Aflatoxins
have been found in soil as well as in grains, nuts, dairy products, tea, spices
and cocoa, as well as animal and fish feeds (Gowda et al., 2012).
Aflatoxin causes
haematological, biochemical, physiological changes in the liver and growth
depression in animals (Gowda et al., 2012). Thus, the presence of mycotoxins in poultry
feeds can result in significant economic losses to the poultry industries. Mycotoxins
have been detected in various food commodities from many parts of the world and
are presently considered as some of the most dangerous contaminants of food and
animal feeds. Adverse effects of mycotoxins on animal health and production
have been recognized in animals kept under intensive management such as
poultry, swine and cattle as a consequence of the consumption of high levels of
contaminated cereals and oilseeds in the diet.
1.1 Statement
of the Research Problem.
Mycotoxins that occur in food and/or
feedstuffs have great significance in the health of humans and livestock (Tola
and Kebede, 2016). The growth of this heterologous group of fungi in feed and
the generation of secondary metabolites of mycotoxins have adverse effects on the
poultry industry and human health as well (Monson et al., 2015; Oliveira
et al., 2015). Exposure of Pregnant women to aflatoxin may cause
increased maternal mortality and low birth weight. Infants exposed to
aflatoxin-contaminated foods may be more susceptible to stunting growth and
malnutrition (Shuaibu et al., 2010).
It has been pointed out that aflatoxin contamination of feeds of
food-producing animals can result in residues of ingested aflatoxins or its
metabolites in meat, milk and egg (Gizachew et al., 2016).
Mycotoxins affect nutritional and economic
value of staple foods and cash crops especially in developing countries
including those in Africa. Of the many mycotoxins, aflatoxin is of major
concern especially countries where agricultural practices are not strictly
controlled, human and animal exposure to mycotoxins is very high (Wagacha et
al., 2014). Attention is only paid to meet export criteria while the
effects of aflatoxin on health of the local consumers is not prioritized. The
contamination of foods with aflatoxin has in recent times created a great alarm
on food security in Africa. In Nigeria,
millet, groundnut, maize and other cereals and legumes are sold in the open
market with less or no regulation of quality. Most of the contaminated foods
find their way into households and restaurants and patronized by unsuspecting
consumers. The assessment of the levels of aflatoxin in food crops and the
identification of fungi responsible for their contamination will inform policy
makers to improve upon proper handling to reduce the toxin in foods.
1.2 Aims and objectives of the study
1.2.1 Aim of the study;
To evaluate
aflatoxin-producing fungi from stored millet cereals.
1.2.2 The objectives of this study;
I.
To isolate common fungi
in harvested millet.
II.
To identity the
aflatoxin-producing fungi isolates in the millet using Thin layer
chromatography.
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