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INHIBITION OF AFLATOXIN PRODUCTION BY ASPERGILLUS FLAVUS ISOLATED FROM STORED RICE USING PLANT EXTRACTS
ABSTRACT
The leaf extracts of Pinus caribaea and Pentaclethra macrophylla were tested for antifungal and antimycotoxigenic activity against the growth of aflatoxin production by Aspergillus flavus using agar dilution method. The results showed that two extracts reduced the growth of aflatoxin production by Aspergillus flavus. Aspergillus flavus was significantly inhibited by the treatment when compared to the control. Further studies should be carried out to know the phytochemicals that are responsible for this antifungal activities.
TABLE OF CONTENT
Title Page i
Certification ii
Dedication iii
Acknowledgment iv
Abstract v
Table of Content vi
List of Tables viii
List of Plates ix
CHAPTER ONE: INTRODUCTION
Introduction 1
Colonization of Mycotoxigenic Fungi on Rice Grains 3
1.3 Causes of Aflatoxin growth in stored rice 5
CHAPTER TWO: LITERATURE REVIEW
2.1 Causes of Aflatoxin Growth in Stored Rice 7
2.2 Site of Infection of Mycotoxin 7
2.3 Aflatoxin Levels in Rice Grains 10
2.4 Management of Mycotoxigenic Fungi Using
Botanical Method 11
2.5 Extraction of Aflatoxin B1 from Aspergillus Flavins
Strains Grown on Rice Grains 14
2.6 Aflatoxin B1 Production by Aspergillins Flavus Isolates
on Rice Grain 15
CHAPTER THREE: MATERIALS AND METHODS
3.1 Collection of plant materials 17
3.2 Sources of Fungal Pathogen 17
3.3 Preparation of plant extracts 18
3.4 Antifungal activity test 18
3.5 Antimycotoxin/mycotoxin activity test of
Aspergillus flavus 19
CHAPTER FOUR: RESULTS AND DISCUSSION
4.1 Fungal Strain 20
4.2 Antifungal Activity Test 23
4.3 Antimycotoxin/Mycotoxin Activity Test 23
4.4 Discussion 24
CHAPTER FIVE: SUMMARY AND CONCLUSION
5.1 Summary 26
5.2 Conclusion 27
REFERENCES 28
LIST OF TABLES
Table 1: Antifungal activity of Pinus Caribaea and
Pentaclethra Macropylla extracts against
mycotoxigenic fungal strain (Aspergillus flavus) 21
Table 2: Antimycotoxin/mycotoxin activity of PinusCaribaeaand
Pentaclethra Macropylla extracts treatment on aflatoxin production
by Aspergillus flavus 22
LIST OF PLATES
Plate 1: Photomicrography of Aspergillus flavus × 400 showing
its conidia and spores. 20
CHAPTER ONE
Introduction
Mycotoxins occurring in food commodities are secondary metabolites of filamentous fungi, which can contaminate many types of food crops throughout the food chain (Reddy et al., 2010). Although, hundreds of fungal toxins are known, a limited number of toxins are generally considered to play important roles in food safety (Shephard, 2008; Reddy et al., 2010). Fungal toxins of most concern are produced by species within the genera of Aspergillus, Fusarium and Penicillium that frequently occur in major food crops in the field and continue to contaminate them during storage, including cereals and oilseeds. Among these mycotoxins, aflatoxin Bl (Arol), fumonisin BI (FBI) and ochratoxin A (OTA) are the most toxic to mammals, causing a variety of toxic effects including hepatotoxicity, teratogenicity and mutagenicity, resulting in diseases such as toxic hepatitis, hemorrhage, oedema, immunosuppression, hepatic carcinoma, equine leukoencephalomalacia (LEM), esophageal cancer and kidney failure (Donmez-Altunta et al., 2003; Santos et al., 2001). The AFBI has been classified as a class I human carcinogen, while FBI land OTA have been classified as class 2B (probable human) carcinogens by the International Agency for Research on Cancer (IARC, 20033). Several outbreaks of mycotoxicoses diseases in humans and animals caused by various mycotoxins have been reported after the.’ consumption of mycotoxin contaminated food and feed (Reddy and Raghavender, 2007).
Several strategies are used at controlling fungal growth and the mycotoxin biosynthesis in stored grains by chemical treatments with ammonia, acids and bases or with food preservatives by physical methods and by biological methods. These methods require sophisticated equipment and expensive chemicals or reagents. Use of natural plant extracts provides an opportunity to avoid chemicals or reagents chemical preservatives. Over the years, efforts have been devoted to search for anti-fungal materials from natural sources for food preservation (Galvano et al., 2001). Several edible botanical extracts have been reported to have antifungal activity (Reddy et al., 2009). The essential oils extracted from clove have been shown to possess significant antifungal properties (Reddy et al., 2007). The inhibitory effects of neem plant extracts on mycotoxin biosynthesis have been examined (Reddy et al., 2009). However, this study will review the developments in control mycotoxigenic fungi and mycotoxins using plant extracts and plant oils to fill the existing gaps and to develop effective antimycogenic natural products for reduction of mycotoxigenic fungi and mycotoxins in foods.
Rice (Oryza sativa) is the most important food crop in Nigeria and the bulk of rice is grown in wet season. Heavy rainfall and floods, particularly near harvest, in coastal areas in eastern, southern, and western regions of the country wet the crop and make panciles more prone to invasion by Aspergillus flavus specie. Reddy et al (2004) in a preliminary study, Aspergillus flavus isolated from rice grains were shown to possess the ability to produce Aflatoxin BI (Reddy et al., 2005). However, mycotoxin producing fungi is les commonly reported for rice than for many cereal a crops (Tanaka et al., 2007). But rice represents a very good substrate for fungal growth since it is used as an ideal culture medium to test the toxigenic potential of isolated strains (Bars, 1992). Among the aflatoxins, aflatoxins BI is the most toxic form for mammals and presents hepatotoxic, teratogenic and mutagenic properties, causing damage such as toxic hepatitis, edema, immunosuppression (Sperijers and Speijers, 2004).
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. The major mycotoxigenic fungi in rice are Aspergillus flavus spp (Reddy et al., 2004), fusarium spp (Abhas et al., 2007). The harmful effects of such fungal invasion are grain discoloration, toxin contamination, loss in quality and viability.
Mycotoxin contamination often occur in the field prior to harvest. Post-harvest contamination can occur if the drying is delayed and during storage of the crop, if the moisture is allowed to exceed critical values for mold growth. (Reddy, 2004). Delayed harvest in rainy season frequently leads to the grains sprouting on the panicle. The fungi Aspergillus flavus, Aspergillus parasiticus and Aspergillus niger have been reported earlier of which Aspergillus flavus have been identified as the primary quality deterrent, producing aflatoxin contaminated seeds when in storage (Desai and Ghosh, 2003).
1.2 Colonization of Mycotoxigenic Fungi on Rice Grains
Discolored grains are frequently encountered in marketed rice and they reduce quality and price. To detect the extent of fungal invasion of kernel starch, discolored kernels were inspected by scanning electron microscopy (SEM) (Mangala et al., 2006). These experiments showed the presence of Aspergillus sp. In kernels of rice cultivars that had invaded starch, endosperm. And embryo This is the first report on the fungal invasion of Aspergillus sp. in rice kernel as studied directly using SEM.
Mycock et al. (1990) studied Aspergillus flavus var. columnaris causing maize seedling infection using SEM. They showed that the fungus was able to invade the internal tissues and observed the hyphae in the xylem of the peduncle of maize seed. Similarly Klich et al. (2000) reported the presence of hyphae of
Aspergillus sp. in the xylem of the outer pigment layer of cottonseed. Balajee et al. (2005) made phenotypic analyses of A. fumigatus on different media using differential interference contrast microscopy and SEM images. Ma et al. (2005) studied the surface ultrastructure and elasticity in growing tips and mature regions of Aspergillus hyphae under atomic force microscope (AFM) and cryo-SEM. Most of these studies on cereal grain infection by Aspergilli were either made by artificial inoculations or by using in vitro cultures.
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