Prevalence of Mycotoxigenic Fungi and Mycotoxins (Ochratoxin A) in Dried Cocoa Beans

Abstract

Concerns about levels of Ochratoxin A (OTA) detected in cocoa beans, its products, and the associated health issues with OTA contamination in cocoa is on the increase. This calls for the need for cocoa producing countries to research and obtain more data and information on the prevalence of Ochratoxigenic fungi present and OTA levels in dried cocoa beans, the raw material for chocolate and other cocoa products. This work sought to determine the moisture content of cocoa beans sampled from farm gates/ societies from licensed buying companies (LBC’s), determine the fungal loads and the occurrence of Ochratoxigenic fungi present as well as the Ochratoxin A levels in the cocoa beans sampled from four cocoa producing districts of the Central Region of Ghana; Agona Swedru, Assin Bereku, Assin Fosu and Breman Asikuma. Hundred (100) samples of dried cocoa beans were obtained from twenty (20) licensed buying companies (Five societies from each LBC). Moisture content of cocoa beans sampled was determined by The International Organization for Standards (ISO) method for moisture content determination. Pour plate method was used in isolating the fungi after surface sterilization with 0.6 % sodium hypochlorite. Fungal identification was done using morphological characteristics such as spore size, shape, and structure as well as colour, using Leica D500 microscope connected to Leica Application Suite (LASEZ) version 2.1. Ochratoxin extraction and quantification by solvent extraction and HPLC-FLD respectively. The method of extraction was partially modified and validated. The limit of detection (LOD) was found to be 1ng/Kg and the limit of quantification (LOQ) was 2ng/Kg. A linear range from 1.0 -20 ng/mL and a correlation coefficient (R2) of 0.9997 were obtained for the entire range of studied concentrations. Repeatability (RSDr) and reproducibility (RSDR) expressed as RSD were 5.5% and 9.8% respectively with mean recovery of OTA spiked at 2.5 and 5ng/kg in 10 replicates were consistent and more than 90%. Cocoa beans obtained from Unicom in the Assin Fosu district recorded the lowest moisture content value of 7.05% and the highest moisture content of 9.86% for the cocoa beans obtained from Nyonkopa in the Assin Bereku district. There were significant differences (P≤ 0.05) among the moisture content recorded for the cocoa beans obtained from the various LBCs. Fungal isolation and identification revealed the presence of fifteen (15) species belonging to eleven (11) genera isolated on Malt Extract Agar and Potato Dextrose Agar from cocoa beans obtained from the four districts sampled. The following species were encountered; Aspergillus (A. flavus, A. fumigatus, A. niger, A. parasiticus), Cladosporium (C. macrocarpum, C. sphaerospermum), Absidia corymbifera, Alternaria alternata, Byssochlamys nivea, Eurotium herbariorum, Mucor racemosus, Penicillium citrinum, Rhizopus stolonifer, Syncephalastrum racemosum and Talaromyces flavus. More species were isolated on Malt Extract Agar compared to Potato Dextrose Agar. Byssochlamys nivea and Talaromyces flavus were not isolated on PDA whilst Mucor racemosus, was also not isolated on MEA. Although all the cocoa beans obtained from the twenty (20) LBCs in the Agona Swedru, Breman Asikuma, Assin Fosu and Assin Bereku districts each recorded the presence of A. niger. Ochratoxin A was not detected in any of the cocoa beans obtained from these sources. Data obtained in this study indicated that most of the cocoa beans were not well dried to the recommended level of between 7% to 8% moisture content. Although some few species of fungi were isolated on both PDA and MEA and identified, only one potential Ochratoxigenic fungu, A. niger was identified and Ochratoxin A was not detected in any of the cocoa beans samples from the four districts. This may be due to the absence of the conditions necessary for the growth of fungi and development of OTA.