A Study on Ochratoxin a and Toxigenic Fungi on Coffee and Selected Cereal Grains Consumed In Ethiopia
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Date
2017-02
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Addis Ababa Universty
Abstract
Ochratoxin A (OTA) is toxic fungal secondary metabolite produced by some filamentous
fungi belonging to the genus Aspergillus and Penicillium. This mycotoxin is commonly
found in coffee and cereal grains and total removal of the toxin by various food processing
methods is not possible. The aim of this research is to study ochratoxin A and toxigenic
fungi in coffee and cereal grains. This dissertation contains five major chapters in which
background information, the rationale for the study and research objectives are presented in
the first chapter. The second chapter is dedicated to reviewing the available literature on the
properties of OTA, the producing fungi, occurrence of OTA in foods, biosynthesis of OTA,
analytical methods, toxicity in human and animals, mechanisms of action and prevention
strategy. This section also addresses production and processing of coffee and some selected
cereal grains. The remaining three chapters are the main experimental part of this study. The
third chapter focuses on the occurrence of toxigenic fungi and OTA in Ethiopian coffee for
local consumption. In this chapter, fungal incidences in coffee samples and OTA
contamination levels in coffee were evaluated using various mycological techniques and
ELISA method for toxin analysis. The identities of fungal isolates were revealed using
species-specific primers and DNA sequencing techniques. The result indicated that total
fungal incidence mounted up to 87%. The predominant fungal genera were Aspergillus (79
%), Fusarium (8 %) and Penicillium (5 %). Ochratosin A producing species of A.
westerdijkiae, A. ochraceus, A. melleus, and A. steynii were identified for the first time using
molecular techniques from locally sold coffee in Ethiopia. A. westerdijkiae and A. ochraceus
were found to be the predominant OTA producers. The median OTA level in the locally sold
Ethiopian coffee was 1.53 μ g/kg. Although samples from wet processing resulted in low median levels of OTA, they were not significantly different from other processing types (p <
0.306). Significant differences in fungal incidences were observed between the different
coffee processing types (p < 0.001), coffee sample types (p < 0.005), and storage materials
(p < 0.03). An in vitro assessment of OTA and Aflatoxins (AFs) production potential of
all Aspergillus isolates reviled that A. westerdijkiae isolates were clearly the most potent
producers of OTA while AFs were only produced by two A. flavus isolates. This chapter is
published in Food Control 69 (2016) 65-73. The fourth experimental chapter deals with the
association of coffee bean defects with Ochratoxin A contamination in the beans: A possible
implication for visual judgment of OTA contaminated coffee beans. In this chapter coffee
bean defects were investigated based on Ethiopian coffee quality and liquoring manual and
Specialty Coffee Association of American (SCAA). The predominant coffee bean defects
encountered in this study in descending order were black beans (47.5 %), cherry (13.5 %),
immature bean (8.1 %), sour bens (7.9%), insect infested (7.9 %), moldy beans (4.4 %), and
broken beans (3.7%). Ochratoxin A contamination levels in coffee beans were positively
correlated with black beans (r = 0.33, p < 0.03), visible mold overgrowth (r = 0.80, p <
0.00), beans damaged by insects (r = 0.71, p < 0.00), and presence of husk (r = 0.67, p <
0.00). These types of defects could be used as a possible visual marker for OTA presence in
locally sold coffee. The fifth chapter deals with High Performance Liquid Chromatography
Variable Wavelength Detector (HPLC-VWD) method of detection and quantification of
Ochratoxin A in teff (Eragrostis teff) and wheat (Triticum spp.) flour samples intended for
local consumption. In this section, HPLC method was developed and validated to detect and
quantify OTA in teff and wheat flour samples. Based on this validated method teff and
wheat four samples were analyzed for OTA contamination using HPLC-VWD apparatus. The results indicate that 20% of the teff and 50 % of the wheat flour samples were
contaminated with median OTA level of 0.99 μg/kg (LOD = 0.78) and 6.76 μg/kg (LOD =
0.58 μg/kg) respectively. This validated method for OTA detection and quantification in
teff and wheat using HPLC-VWD detector generates meaningful data that satisfy
performance criteria set by European Commission, EC 401, 2006 and Eurachem Guideline
validation requirements. Moreover, an insight into the occurrence of OTA in teff is very
valuable because the cereal might provide a low OTA risk alternative for wheat in Ethiopia.
Keywords: Aspergillus spp., Aflatoxins, toxigenic fungi, coffee, defect count, mycotoxin,
ochratoxin, ELISA, HPLC-VWD, teff, wheat
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Keywords
Aspergillus spp, Aflatoxins, Toxigenic fungi, Coffee, Defect count, Mycotoxin, Ochratoxin, ELISA, HPLC-VWD, Teff, Wheat