ISOLATION AND IDENTIFICATION OF FUNGI PRODUCING MYCOTOXINS FROM RICE GRAINS SOLD IN UMUAHIA MARKETS

ABSTRACT

The present research work explores fungi and mycotoxins associated with rice grains. Mycotoxins are secondary metabolites of molds that have adverse effects on humans, animals and crops that result in illnesses and economic losses. Total of 18 rice samples collected from different markets in Umuahia (Ubani, Orieugba and Ndioru) were analyzed. Six different fungi were isolated altogether namely; Aspergillus niger, Aspergillus flavus, Penicillium sp., Rhizopus sp., Fusarium sp. and Yeast sp. In the result for the percentage occurrence of fungal isolated in rice samples Yeast sp. accounted for 83.3%, followed by Aspergillus niger (50%), Aspergillus flavus and Penicillum sp. (38.9%), Rhizopus sp. (22.2%) and Fusarium sp. (16.7%). Mycotoxin production by isolated fungi was subsequently evaluated using the Thin Layer Chromatography (TLC) technique. In the results, mycotoxin assay revealed that only Aflatoxin was produced in this study. Not all fungi produced toxin. While Aspergillus flavus and A.niger produced Aflatoxin, Fusarium sp., Rhizopus sp., Penicillium sp., and Yeast sp., did not produce any toxin. The result of fungal load demonstrated that Mars rice brand from Ubani market recorded the highest count with 54 x 102 cfu/g, samples from Orieugba market showed that Royal stallion rice has the highest count of 37 x 102 cfu/g and Mars rice from Ndioru market equally recorded a high count (46 x 102 cfu/g) at P<0.05, this was a significant difference in the fungal count from samples of various markets. Detection of fungi and aflatoxins in rice poses a risk for consumer’s health owing to the fact that rice is highly consumed and it is necessary to check the rice grains before allowing distribution for public use.

TABLE OF CONTENTS

Dedication                                                                                                                              i

Acknowledgement                                                                                                                  ii

Table of Contents                                                                                                                   iii-v

List of Tables                                                                                                                          vi

Abstract                                                                                                                                  vii

CHAPTER ONE

1.0           Introduction

1.1       Background of the Study                                                                                            1-2

1.2       Significance of the Study                                                                                           2

1.3       Aim of the Study                                                                                                        3

1.4       Objective of the Study                                                                                                3

CHAPTER TWO

2.0           Literature Review      

2.1       Historical Review of the Study                                                                                  4-5

2.2       Nature of Mycotoxins                                                                                                 5

2.3       Types of Mycotoxins                                                                                                  5-11

2.4       Classification of Mycotoxin-producing Fungi                                                           12

2.5       Routes of Contamination                                                                                           13

2.6       Site of Infection of Mycotoxigenic Fungi in Rice Grains                                          13-15

2.7       Mycotoxigenic Fungi in Rice                                                                         15-17

2.8       Strategies for Prevention of Mycotoxin                                                                     17-18

2.9       Control Measures                                                                                                        18-20

CHAPTER THREE

3.0           Materials and Methods

3.1       Sources of Materials                                                                                                   21

3.2       Sampling and Sample Preparation                                                                             21

3.3.0    Isolation of Fungi                                                                                                       22

3.3.1    Characterization of Fungi Isolates                                                                              22

3.3.2    Identification of Fungi Isolates                                                                                  22

3.4.0    Screening of Rice for Mycotoxins                                                                              22-23

3.4.1    Thin Layer Chromatography (TLC) Method for Screening of Mycotoxins                 23-24

CHAPTER FOUR                 

4.0           Results                                                                                25

CHAPTER FIVE

5.0       Discussion, Conclusion and Recommendation

5.1       Discussion                                                                         30-32

5.2       Conclusion                                                                                33

5.3       Recommendation                                                                                                       33-34

References

Appendix I

Appendix II

Appendix III

Appendix IV

Appendix V

LIST OF TABLES

Table 1 Colony and Structural Features of the Fungi Isolated           26

Figure 1: Percentage Occurrence of Fungal Isolates in Rice Samples          27

Table 2 Fungal Load of the Fungi Isolated                                          28

Table 3   Statistical Analysis of Fungal Load of the Fungi Isolated   29

CHAPTER ONE

1.0       INTRODUTION

            1.1           Background of the Study

It is difficult to define mycotoxin in a few words. All mycotoxins are low-molecular-weight natural products (i.e., small molecules) produced as secondary metabolites by filamentous fungi. These metabolites constitute a toxigenically and chemically heterogeneous assemblage that are grouped together only because the members can cause disease and death in human beings and other vertebrates. Mycotoxins are toxic substances produced mostly as secondary metabolites by filamentous fungi that grow on seeds, grains, and feed in the field, or in storage. The term ‘mycotoxins’ is produced by fungi that grow on crops and variety of feed and foodstuffs. One of the most important effects of post harvest decays of seed and feed deterioration by fungi is the induction of mycotoxicoses. This is a disease of animals and humans following consumption of feeds and foods invaded by fungi that produce toxic substances called mycotoxins (Agrios, 1978; Moss, 1989). Mycotoxins are natural contaminants of cereals and other food commodities throughout the world and they significantly impact human and animal health.

Rice is a cereal grass (Oryza sativa) that is cultivated extensively in warm climates for its edible grains. Rice is an important food crop worldwide along with wheat and corn, and has been major food in several countries. Rice is not spared from natural mycotoxin contamination as bulk of it is grown in kharif or the wet season. Frequent and heavy rainfall and floods, particularly near harvest, in coastal are as in Eastern, Southern, and Western regions of the country wet the crop and make panicles more prone to invasion by fungi and bacteria.

During the wet season, sun drying practiced by most farmers may not adequately reduce the moisture content in grains. Thus, rice grains with moisture content higher than the desired level enter the storage system. As a result, invasion by both field and storage fungi takes place. Therefore, Mycotoxin - producing moulds could contaminate the grain and produce important quantities of mycotoxins during storage. Climate and storage conditions have been registered to have influence on the formation of the intended mycotoxins.

The major mycotoxigenic fungi in rice are Aspergillus sp., Fusarium sp. and Penicillium sp. Aflatoxins (AFs) are a group of mycotoxins produced as secondary metabolites by the spoilage of Aspergillus fungi, particularly Aspergillus flavus and Aspergillus parasiticus (Davis and Diener, 1983; Miguel and Guillermo, 1986; Yu et al., 2003; Klich, 2007). These fungi can grow on various agricultural commodities and generate aflatoxins before and during harvest, handling, shipment and storage (Peraica et al., 1999; Giray et al., 2007; Reddy et al., 2009a). Several mycotoxicoses in humans and animals have been reported due to the consumption of mycotoxin-contaminated food and feed (Peraica and Domijan, 2001; Reddy and Raghavender, 2007).

            1.2           Significance of the study

Microorganisms propagules get on grain in different ways, most often with dust from soil, from the surface of plant remnants during harvesting, transportation, storage and processing. The simultaneous occurrence of highly toxic mycotoxins often represents a major obstacle in the isolation of vital antibiotic and antifungal agents. Therefore, their presence as well as the occurrence of mycotoxins should be further investigated to assess health risks.

1.3       Aim of the study

The scope of the study is based on the screening of fungi isolated from rice for the occurrence of mycotoxins which causes mycotoxicoses.

1.4        Objective of the study

     a.         To isolate and identify fungi from different rice brands sold in Umuahia markets.

     b.         To test the isolated fungi for mycotoxin production.

     c.         Determination of mycotoxins in rice.

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