ABSTRACT
Aflatoxins are natural toxigenic metabolites frequently found in groundnut, millet and cereal products. Asperigillus species are mainly responsible for aflatoxin buildup and contamination of groundnut products. The main objective is to detect aflatoxin-producing moulds in groundnut commonly sold in Ndoru Market Umuahia,Abia state four (4) groundnut samples were bought from four different sales point in Ndoru Market from which (4) organisms were isolated using spread plate techniques and belong to 2 genera and 4 species. Asperigillus flavus,Asperigillus spp, Penicillium spp. The percentage of occurrence is Asperigillus flavus was found in (47%), Asperigillus spp (47%), and Penicillium spp (25%), Penicillium spp (4%).However the highest prevalence of occurrence was seen in A.flavus. Aflatoxin production by isolated fungi was subsequently evaluated using the thin layer chromatography technique and viewed under UV light. Two isolates Asperigillus flavus, Asperigillus spp of Asperigillus genera produced green fluorescence as detected in the culture filtrates while the remaining two isolates Penicillium sp of produced no fluorescence. The green fluorescent colouration emitted under UV light which indicates the ability to produce aflatoxin by aflatoxigenic strains was not observed from any of the tested non-aflatoxigenic isolates. The findings from this study indicates that groundnut obtained from the two (2) out of the four (4) different sales point were 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
Dedication iii
Acknowledgements iv
Table of Contents v
Lists of Tables viii
Abstract
ix
CHAPTER ONE
1.0 Introduction 1
1.1 Aim and Objectives 6
1.2 Objectives 7
CHAPTER TWO
2.0 Literature Review 8
2.1
Overview of Aflatoxin 8
2.2.1 History of Aflatoxin 8
2.2.2 Types of Aflatoxin 9
2.3 Mycotoxin 12
2.3.1 Types of
Mycotoxin 13
2.3.1.1 Aflatoxin 13
2.3.1.2 Ochratoxins 14
2.3.1.3 Fumonisins 14
2.3.1.4 Zearalenone 15
2.4
Global Occurrence and Spread of Aflatoxin
16
2.5
Incidence Of Aspergillus Contamination
of Groundnut (Arachis hypogaea L.) 18
2.6 Factors Influencing Fungal Infection
and Aflatoxin s Contamination in Food Materials 21
2.6.1. Climatic factors 21
2.6.2. Agronomic and biotic factors 22
2.6.2.1. Drying 22
2.6.2.2. Storage 23
2.7 Methods for Isolation
and Identification of Aspergillus 24
2.7.1 Microbiological isolation and identification methods 24
CHAPTER THREE
3.0
Materials and Methods 27
3.1 Sample Collection 27
3.2
Media 27
3.3
Sterilization 27
3.4
Isolation of Aspergillus from Groundnut 27
3.5 Screening of Aflatoxigenic Aspergillus
spp. 27
3.6 Determination of Aflatoxin Using
Thin Layer Chromatography (TLC) Technique 28
3.6.1 Method for TLC 28
3.6.2 Calculation of Retention Factor
(RF) 29
3.7
Characterization of Fungi in Groundnut 29
CHAPTER FOUR
4.0
Results 30
CHAPTER FIVE
5.0 Discussion, Conclusion and
Recommendation 34
5.1 Discussion 34
5.2 Conclusion 34
5.3
Recommendation 34
References 37
Appendix
LIST OF
TABLES
Tables Title
Page
4.1 Morphological
And Microscopic Examination Of Fungi Isolated from
Groundnut Samples From
Different Sales Point In Ndoru Market. 31
4.2 Occurrence
of Isolates from Groundnut Samples 32
4.3 Identification of Aflatoxin Producing Fungi by Their
Fluorescencing Characteristic. 33
CHAPTER ONE
1.0
INTRODUCTION
Mycotoxins are natural contaminants in raw materials, food and
feeds (Bosco and Mollea, 2012). They are toxic metabolites produced by
different species of toxigenic fungi, especially by saprophytic moulds growing
on foodstuffs or animal feeds. Not until the past 30 years, the effects of
these moulds and their toxins have been largely overlooked and have been
sources of hazard to man and domestic animals. Although poisonous mushrooms
have been carefully avoided, moulds growing on foods have generally been
considered to cause unaesthetic spoilage, without being dangerous to health
(Abdel-Gawad and Zohri, 2013; Ahmad, 2013). Between 1960 and 1970 it was
established that some fungal metabolites, now called mycotoxins, were
responsible for some animal diseases and death (Blount, 2013). In the decade
following 1970, it became clear that mycotoxins have been the cause of human
illness and death as well (Alpert et al, 2013; Richard, 2012).
The discovery of Aflatoxin (AFs)
dates back to the year 1961 following the severe outbreak of turkey X disease,
in England, resulting in the deaths of more than 100,000 turkeys and other farm
animals (Quist, et al., 2012). The cause of the disease was attributed
to a contaminated feed. Thin-layer chromatography (TLC) revealed that a series
of fluorescent compounds, later termed Aflatoxin, were responsible for the
outbreak (De-Iongh et al., 2012; Balzer et al., 2017). The
disease was linked to a peanut meal, incorporated in the diet, contaminated
with a toxin produced by the filamentous fungus Aspergillus flavus. Hence,
the name Aflatoxin, an acronym that was formed from the following combinations:
the first letter, A for the genus Aspergillus, the next set of three
letters, FLA, for the species flavus, and the noun Toxin meaning poison
(Rustom, 2017).
Aflatoxin (AFs) are difuranocoumarins produced
primarily by two species of Aspergillus fungus which are especially
found in areas with hot, humid climates (Criseo et al., 2013; Udom et
al., 2012). Aspergillus Section flavi contains a number of
species capable of producing a wide array of mycotoxins among which Aflatoxin are
the most important in food safety. Aflatoxin
are potent carcinogenic, mutagenic, and teratogenic secondary metabolites and
are produced predominantly by Aspergillus flavus and Aspergillus
parasiticus (Bennett and Papa, 2017). There exists basically two groups of Aspergillus,
the Aflatoxin -producing species such as Aspergillus flavus, A.
parasiticus, A. nomius, A. flavus and the recently described species, A.
pseudotamarii and A. bombycis (Cary and Ehrlich, 2014). The other
group includes the Aflatoxin non-producing species: A. oryzae, A. sojae, and
A. tamarii, which have been used for production of traditional fermented
foods in Asia (Kumeda and Asao, 2013).
Aflatoxin belong to the class of mycotoxins (Huang et al., 2010). Chemically
they are defined as difuranocyclopentano-cumarines or difuranopentanolido-cumarines
that is Aflatoxin containing a
dihydrofuran or a tetrahydrofuran ring, to which a substituted cumarin system
is condensed. Out of about 20 known Aflatoxin
, the moulds Aspergillus flavus and
A. parasiticus produce exclusively Aflatoxin B1, B2, G1 and G2, and all the other Aflatoxin are derivates of these four (Arseculeratne et
al., 2014; Huang et al., 2010). The derivates are developed either by
metabolism in humans, animals and microorganisms or by environmental reactions.
Among the 18 different types of Aflatoxin identified, the major members are Aflatoxin
B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2), M1 (AFM1) and M2 (AFM2). Aflatoxin B1
is normally predominant (in amount) in cultures as well as in food products
(Arseculeratne et al., 2014).
Pure AFB1 is a pale-white to yellow crystalline, odorless solid. Aflatoxin are soluble in methanol, chloroform,
acetone and acetonitrile (Asao et al., 2013).
Aspergillus flavus typically
produces AFB1 and AFB2, whereas A. parasiticus produce AFG1 and AFG2 as
well as AFB1 and AFB2 (Huang et al., 2010). Four other Aflatoxin M1, M2,
B2A, G2A which may be produced in minor amounts were subsequently isolated from
cultures of A. flavus and A. parasiticus (Gulyas, 2016; Huang et
al., 2010). A number of closely related compounds namely Aflatoxin GM1, parasiticol and aflatoxicol are
also produced by A. flavus (Nesbit et al., 2012; Gulyas, 2016).
The order of acute and chronic toxicity produced by these Aflatoxin is
AFB1 > AFG1 > AFB2 > AFG2. The degree of the severity is therefore
reflecting the role played by epoxidation of the 8, 9-double bond and also the
greater potency associated with the cyclopentenone ring of the B series, when
compared with the six-membered lactone ring of the G series. Aflatoxin M1 and M2 are hydroxylated forms of
AFB1 and AFB2 (Dors et al., 2013).
AFM1 and AFM2 are major metabolites of AFB1
and AFB2 in humans and animals and may be present in milk from animals fed on
AFB1 and AFB2 contaminated feed (Gundinc and Filazi, 2014; Filazi et al.,
2010). Furthermore,
it may also be present in poultry eggs (Zaghini et al., 2016), corn
(Shotwell et al., 2016) and peanut (Ren et al., 2018; Huang et
al., 2010). Aflatoxin interact
with the basic metabolic pathways of the cell disrupting key enzyme processes
including carbohydrate and lipid metabolism and protein synthesis (Quist et
al., 2012). The health effects of Aflatoxin have been reviewed by a number of workers (Tang, 2016; Kensler et
al., 2012; Pang et al., 2016). Aflatoxin are among the most potent
carcinogenic, teratogenic and mutagenic compounds in nature (Shephard, 2016;
Kirk et al., 2014; Jackson and Al-Taher, 2017). The International Agency
for Research on Cancer (IARC) has concluded that naturally occurring Aflatoxin belong to group 1 carcinogens to humans,
with a role in the aetiology of liver cancer, notably among subjects who are
carriers of hepatitis B virus surface antigens. In experimental animals, there
was sufficient evidence for carcinogenicity of naturally occurring mixtures of Aflatoxin
and of AFB1, AFG1 and AFM1, limited evidence for AFB2 and inadequate evidence
for AFG2. The principal tumours were in the liver, although tumours were also
found at other sites including the kidney and colon. Aflatoxin B1 is consistently genotoxic in vitro
and in vivo (EFSA, 2018).
The Joint Food and Agriculture Organization of the United Nations
(FAO)/World Health Organization (WHO) Expert Committee on Food Additives
(JECFA) concluded that AFM1 should be presumed to induce liver cancer in
rodents by a similar mechanism to AFB1, and that estimates of the potency of
AFB1 can be used for determining the risk due to intake of AFM1, including
those for populations with a high prevalence of carriers of hepatitis B virus.
The carcinogenic potency of AFM1 was estimated to be one-tenth that of AFB1,
based on a comparative study in the Fischer rat conducted by Cullen et al.
(2018). Humans can be exposed to Aflatoxin by
the periodic consumption of contaminated food, contributing to an increase in
nutritional deficiencies, immunosuppression and hepatocellular carcinoma. Aflatoxin have a wide occurrence in different kind of
matrices, such as spices, cereals, oils, fruits, vegetables, milk and meat
among others (Dors et al., 2013).
About 4.5 billion people, mostly in developing countries, are at
risk of chronic exposure to Aflatoxin from contaminated food crops (Shuaib et
al., 2017). Therefore, in order to avoid the toxicity, the levels of Aflatoxin
and similar toxic compounds in
foodstuffs have to be monitored closely, and to be kept under control
continuously. Otherwise, related health effects like acute and chronic
intoxications, and even deaths, will still be an issue (Becer and Filazi,
2010). Mycotoxins can be acutely or chronically toxic, or both, depending on the
kind of toxin and the dose (Richard, 2012; Kensler et al., 2012). Acute mycotoxicoses
include among others ergotism, a condition caused by a metabolic product known
as ergot produced by Claviceps purpurea, alimentary toxic aleukia (ATA),
a condition caused by a group of mycotoxins known as trichothecene (T-2). Other mycotoxicoses of acute nature include acute
cardiac beriberi caused by citreoviridin,
a mycotoxin produced by the comparatively rare species, Penicillium
citreonigrum (Uragochi, 2013). Onyala is another mycotoxicosis of
acute significance. It was discovered that toxigenic isolates of Phoma
sorghina were found to be common in Groundnut consumed by affected
population (Rabie et al., 2015). Aspergillus flavus is
ubiquitous, favouring the aerial parts of plants (leaves, flowers) and produces
B Aflatoxin. Aspergillus parasiticus which produces both B and G Aflatoxin,
is more adapted to a soil environment and has more limited distribution (EFSA, 2018).
Aspergillus
bombysis, A. ochraceoroseus, A. nomius,
and A. pseudotamari are also AFs producing species, but are encountered
less frequently. From the mycological perspective, there are qualitative and
quantitative differences in the toxigenic abilities displayed by different
strains within each aflatoxigenic species.
For example, only about half of A. flavus AFs-producing strains produce
AFs- more than 106 g kg−1 (Turner et al., 2014). Aflatoxin are secondary metabolites known to be
highly toxic and the most carcinogenic of natural toxins (IARC, 2015). They are
produced by some strains of section flavi (Aspergillus flavus, Aspergillus
parasiticus, Aspergillus nomius, Aspergillus bombycis and Aspergillus
pseudotamarii) and isolates outside this section such as Aspergillus
ochraceoroseus in section circumdati, and Emericella astellata and
Emericella Venezuelensis (Criseo et
al., 2016; Cary and Ehrlich, 2014; Johnsson et al., 2017; Reddy et al.,
2014). This group of fungi has been subjected to detailed investigations. A.
flavus and A. parasiticus are
the main Aflatoxin producing species. They are frequently found in foodstuffs
and animal feeds and are associated with a wide spectrum of stored agricultural
commodities. However, not all Aspergillus species are able to produce Aflatoxin.
Different methods are implemented to screen the ability of Aflatoxin production
of Aspergillus species. These methods commonly use the culture of
strains in suitable liquid or solid media. Aflatoxin produced are then analysed by
chromatographic and ELISA techniques (Lin et
al., 2017; Yang et al., 2015).
For this purpose many media are used: Yeast extract-sucrose (YES) (Fente et al., 2016), Reddy medium, and natural
media with wheat, rice, peanut, malt, date, palm kernel or coconut extracts
(Hara et al., 2014; Ahmed and
Robinson, 2014; Klich, 2015; Atanda et al.,
2014).
To
meet the need for more environmentally sound methods which may be applicable
and available to screen large numbers of strains in a reasonable time,
alternative methods were developed. These are based on the use of complex media
to detect the natural fluorescence of Aflatoxin
released
by the growing mycelium (Hara et al.,
2014; Fente et al., 2013; Maragos et al., 2017) or rely on multiplex
Polymerase chain reaction (PCR) and real time Polymerase chain reaction
(RT-PCR) detection of genes or their transcripts involved in the Aflatoxin biosynthetic
pathway (Färber et al., 2018; Criseo et al.,
2016; Somashekar et al., 2015; Scherm et al.,
2016). New instrumental techniques approaches for Aflatoxin determination
such as fluorescence polarisation, multiphoton-excited fluorescence, LC
separation followed by electrospray ionisation-MS-MS liquid
chromatography-electrospray ionization/multi- stage mass spectrometry
(LC/ESI–MS–MS) detection were also
developed but all these alternative methods are not always available or
affordable to developing countries. In the present study coconut broth has been
used as medium to produce Aflatoxin which
have been analysed by HPLC. A. flavus and A. parasiticus medium
have been used to identify isolates belonging to A. flavus or A.
parasiticus species (Trucksess, 2012).
1.1 Aim and Objectives
The
aim of this study is to determine the presence of Aflatoxigenic Aspergillus isolated in groundnut.
1.2 Objectives
1.
To isolate and identify Asperigillus
in groundnut.
2.
To characterize Aflatoxigenic Aspergillus
isolated in groundnut.
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