OCCURRENCE OF AFLATOXIN PRODUCING FUNGI IN THREE FOODS, RICE (ORYZA SATIVA), MAIZE (ZEA MAYS) AND GROUNDNUT (ARACHIS HYPOGAEA) SOLD IN AND AROUND UMUDIKE, UMUAHIA ABAIA STATE

ABSTRACT

This work was aimed at determining the occurrence of aflatoxin producing fungi in three (3) foods: Rice (Oryza sativa), Maize (Zea mays) and Groundnut (Arachis hypogaea) sold in and around Umudike, Abia State, Nigeria. Samples were randomly collected from sellers in three (3) different markets (Umudike, Ahiaeke and Amawom). Serial diluted samples were plated on saboraud dextrose agar using spread plate methods and incubated at 250C for five (5) days, after which fungal growth and mycotoxin production were evaluated using standard method. Results obtained showed the presence of five (5) fungal species: Aspergillus niger, Aspergillus flavus, Penicillum species, Fusarium species and Yeasts. Fungal isolates showed varied occurrence: Aspergillus niger (88.9%), Aspergillus flavus (66.7%), Penicillum (66.7%), Fusarium (44.4%) and Yeasts (100%). The occurrence of A. niger  in foods showed varying occurrence; rice (66.7%), maize (100%) and groundnut (100%), while A. flavus had 33.3%, 66.7% and100% in seeds respectively. The corresponding values for Penicillum were 33.3%, 100% and 66.7% whereas Fusarium recorded 0%, 33.3% and 100% for the same seeds. Yeasts were present in all the samples (100%). The gross occurrence of aflatoxin detected in foods were:  maize (22.2%), groundnut (22.2%) and 0% in rice. The quantitative analysis shows Aflatoxin content ranged 37.76µg/kg to 60.72µg/kg with mean values of 39.80% in maize and 57.01% in groundnut. The variations in the occurrence of fungi, detection of aflatoxin in the seeds and the concentrations of aflatoxin in the foods were noted and it was observed that consumers of these foods are exposed to potential harmful effects. More aflatoxins were in the groundnut than in the maize and rice. Based on the findings, it was therefore recommended that there should be public enlightenment on the incidence of mycotoxins in our foods presently and the high risk associated with it.

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                               iii

Acknowledgements                                                                                                                iv

Table of contents                                                                                                                    v

List of tables                                                                                                                           vii

List of figures                                                                                                                         viii

Abstract                                                                                                                                   ix

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aim and Objectives                                                                                                    3

CHAPTER TWO

2.0       Literature Review                                                                                                       4

2.1       Aflatoxin Producing Fungi                                                                                         4

2.2       The Health Implications of Aflatoxins in Humans and Animals                             4

2.3       Food Grains that can be Contaminated with Aflatoxin                                              6

2.4       Major Diseases (Disease Classification) Caused by Aflatoxin Consumption  8

2.5       Factors favourable for the Occurrence of Aflatoxins in Food Grains                         10

2.6       Aflatoxin in Contaminated Maize                                                                              11

2.7       Possibilities of Maize Being Contaminated by Aflatoxin Producing Fungi               14

2.8       Aversion of Aflatoxins in Maize                                                                                15

2.9       Aflatoxin Contamination in Groundnut                                                                    17

2.10     Prevention of Aflatoxins in Groundnut                                                                      18

2.11     Aflatoxin in Rice (Oryza sativa)                                                                                19

CHAPTER THREE

3.0       Materials and Methods                                                                                               21

3.1       Source of Materials                                                                                                    21

3.2 Sampling, preparations                                                                                                     21

3.2.1 Sampling and sample preparation                                                                                 21

3.2.2 Media preparation                                                                                                         21

3.3       Isolation of fungi contaminants of the seeds                                                              22

3.3.1. Characterization of fungi isolates                                                                                 22

3.3.2. Identification of fungi isolates                                                                                      23

3.4       Determination of aflatoxin                                                                                         23

3.4.1. Extraction of aflatoxin                                                                                                  23

3.4.2 Preparation of thin layer chromatography plate                                                                        24

3.4.3 Detection of Aflatoxin                                                                                                   24

3.4.4    Determination of aflatoxin content                                                                           25

CHAPTER FOUR

4.0       Results                                                                                                                        28

CHAPTER FIVE

5.0         Discussion, Conclusion and Recommendation                             33

5.1         Discussion                                                                                                                 33

5.2         Conclusion                                                                                                                35

5.3         Recommendations                                                                                                    35

References                                                                                                                  36

LIST OF TABLE

Table              Title                                                                             Page  

1:       Aflatoxin Content of Seed Foods in Umudike                  32

LIST OF FIGURES

Figure                    Title                                                              Page  

1:                     Occurrence of Fungi Isolates                                 29

2:                     Detection of Aflatoxin in Seeds                            30

CHAPTER ONE

 1.0    INTRODUCTION

We are all concerned with the quality and safety of food. Harmful components in plants derived foods may be manmade sources or from the activities of microorganisms. Cereals and legumes are some of the foods consumed in most parts of Africa and the world over (Chauvin et al., 2012). Rich in different nutrients such as carbohydrates, proteins and vitamins, these cereals and legumes are also susceptible to fungal infections both on growing fields and in stores (Piotrowska   et al., 2013; Pereira  et al., 2014). Aflatoxins occurring in food commodities are secondary metabolites of filamentous fungi, they are group of chemically similar compounds that contaminate many crops in hot, humid climates. Aflatoxins are natural contaminants in raw materials, food and feeds (Bosco and Mollea, 2012). They are usually produced as metabolites by these microorganisms (fungi) and are described as mycotoxins.

Aflatoxins (Afs) are produced by the three main genera Aspergillus, fusarium and penicillium during crop growth, harvesting or storage. They are produced primarily by two species of Aspergillus (A.flavus and A. parasiticus) fungus which are especially found in areas with hot, humid climates. These metabolites may not be necessary for the growth and development of the fungi, it could help them to eliminate and compete with other microorganisms in the same environment. Aflatoxins are highly toxic and carcinogenic substance and are produced and deposited in a variety of agricultural products including cereals and some legumes. According to Gorya et al., (2009), these toxins have carcinogenic and mutagenic properties as well as nephrotoxic and hepatotoxic properties which altogether make them harmful to both humans and animals. These fungi affects cereals notably peanuts, corn, wheat and rice. Aspergillus and penicillium were reported as the most dominant genera in the African peanut seeds.

PrH, (2000) reported that the major naturally produce aflatoxins are aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2. A.flavus is ubiquitous favoring the aerial parts of plants (leaves, flowers) and produces B aflatoxins. A.parasiticus produces both B and G aflatoxins, is more adapted to soil environment and has more limited distribution (EFSA, 2007). A. bombysis, A. ochraceoroseus, A. nomius and A. pseudotamari are also aflatoxins producing species, but are encountered less frequently. Aflatoxin B1 is the most common in food and amongst the most potent genetoxic and carcinogenic aflatoxins, it is the most important and toxic to human beings from the public health point of view. It is the most toxic and potent carcinogen, teratogen and mutagen to human and animals (Sweeney and Dabson, 1998; Shahidi, 2004; Seo et al., 2011), causing damage such as toxic hepatitis, hemorrhage, edema, immunosuppression and hepatic carcinoma (Speijers and Speijers, 2004, Peng and Chen, 2009; Woo et al., 2011). Aflatoxin M1 and M2 were also isolated and identified as mammalian metabolites of aflatoxin B1 and B2.

Due to the fact that these crops serve as staple crops and are consumed on a daily basis, concerns have grown recently on the possible occurrence of mycotoxins in these crops and the implications of these mycotoxins on human and animal health. This has led to the introduction of maximum tolerated limits for mycotoxins in several foods and food products in various countries. Developing maximum limits requires information about the toxicity and the occurrence of mycotoxins in food (Wagacha and Muthomi, 2008). Reports show that contamination of cereals with toxic producing microorganisms may occur before during or after harvest especially during storage. Poor hygiene of handlers and their environment in addition of high temperature and moisture are factors that aid microbial contamination of cereal at post-harvest level and there are often the situations in most African settings (Jackson et al., 2008). Researchers and public health personnel are in agreement with the report of Ngeda et al., (2011) that there is need to for mycotoxin surveillance, because of their wide occurrence in contaminated commodities. In line with the above assertions, this project was undertaken to survey the occurrence of aflatoxins in some seed foods sold in and around Umudike (a university community) as one way of determining the potential dangers to which consumers have are exposed.

1.1       AIM AND OBJECTIVES

The aim of this project was to determine the occurrence of aflatoxin producing fungi in three foods, Rice (Oryza sativa), Maize (Zea mays) and Groundnut (Arachis hypogaea) sold in and around Umudike.

The objectives of the study include the following:

1.     To isolate and identify fungi in the three test foods.

2.     To screen the fungal isolates for their ability to produce aflatoxins.

3.     To determine the relative occurrence of the fungi that produce aflatoxin in the foods.

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