Journal of Experimental Biology and Agricultural Sciences, July - 2013; Volume – 1 (3)
Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org
ISSN No. 2320 – 8694
STUDIES ON FACTORS WHICH PREDISPOSE FERMENTED COCOA (Theobroma cacao) BEANS TO MYCOTOXIN CONTAMINATION
Abrokwah FK1, Takramah J2, Ocloo A1 and Sackey ST1* 1 2
Department of Biochemistry, Cell and Molecular Biology, University of Ghana. Cocoa Research Institute of Ghana, P. O. Box 8, New Tafo Akim, Ghana.
Received: June 02, 2013; Revision: June 27, 2013; Accepted: July 08, 2013 Available Online July 20, 2013.
KEYWORDS Pod integrity Sterilization Cocoa beans Ochratoxin A Contamination
ABSTRACT To investigate the effects of pod integrity, pod surface contamination and effective bean drying on the level of Ochratoxin A contamination in dried cocoa beans from Ghana, ripe cocoa pods were harvested at Akim Tafo (Eastern Region) and sorted into four groups: healthy pods, diseased pods, diseased and damaged pods and damaged/broken pods. Each group was subjected to three main recommended fermentation systems, i.e. traditional heap, tray and box. Some of the treatments were replicated in two other ecological zones in Ghana where cocoa is produced viz Assin-Worakese (Central Region) and Wantram (Western Region). Fermented beans were dried in the open sun for a minimum period of nine days during which samples were taken for quantitative analysis of the level of Ochratoxin A using commercially available kit from Helica Biosystem Inc. Ochratoxin A contamination appear to be associated with the integrity (intactness) of the pods from which they were obtained, duration and conditions of drying, farm to farm practices and season of primary processing. Beans obtained from diseased pods and damage/broken pods gave the highest Ochratoxin A levels. None of the levels detected was however in the range considered by the European Union as inimical to human health. Ghana Cocoa Board recommendation to farmers is that damaged and diseased pods should not be fermented with wholesome pods, and that beans should be dried to completion in maximum of seven days. The study shows that if these conditions are adhered to, contamination of Ghana’s Cocoa beans by Ochratoxin A can be entirely prevented.
* Corresponding author E-mail:
[email protected] (Sackey S T) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.
Production and Hosting by Horizon Publisher (www.my-vision.webs.com/horizon.html). All rights reserved.
175
Abrokwah et al.
1 Introduction The contents of an intact cocoa pod/fruit, a pod/fruit, which is not affected by any disease or attacked by any animal is normally sterile and free from microorganism specially fungi contamination and hence mycotoxin contamination. However, when the pod/fruit is infected with diseases, attacked by insects or rodents or injured on the farm prior to harvest or during harvest, the beans are exposed to bacterial and fungal contaminations (Amezqueta et al., 2008). Even though most of these bacterial species (Lactobacillus, Acetobacter and Lactococcus species) and fungal species (Saccharomyces and Hanseniaspora species) (Illeghems et al., 2012) are important in fermentation and the fruit pulp or mucilage of the cocoa seeds offer excellent living conditions for these microorganisms (Schwan 1998), when they get access to the interior of the pods/fruits before or during harvest and if drying is not properly carried out, they get enough time to undergo their normal metabolism and also produce secondary metabolites such as mycotoxins (Mounjouepou et al., 2008). Mycotoxins are mainly low molecular weight compounds produced by Aspergillus, Penicillium and Fusarium species that may contaminate cocoa pods and beans alike (Abarca et al., 2000). Mycotoxins of greatest importance to mycologists are aflatoxins, ochratoxin A, fumonisins, trichothecenes and zearalenone because of their known toxic properties (Pitt 2000). Ochratoxin A (OTA), which is nephrotoxic, immunotoxic, teratogenic and carcinogenic (Petzinger and Weidenbach 2002), is a secondary metabolite produced by several species of Aspergillus and Penicillium genera (Mounjouepou et al., 2008). A study by Amezqueta et al. (2008) has shown that OTA production is likely to be intensive during drying of cocoa beans if conditions are not ideal. Pardo et al. (2004) also suggested that the production of OTA is not a result of storage alone, but is more likely linked to adverse conditions during harvesting and drying. Similarly, Joosten et al. (2001) observed that drying is the most critical point in coffee processing for OTA contamination, hence by extrapolation is also likely to be the most important or critical point for cocoa. Consumer exposure to OTA is reported to be increasing gradually and in order to protect consumers the European Union has drawn up a standard to define tolerable contamination limits (European Commission, 1995). Other products, such as cereals, coffee and wine are already covered by international regulations; however, this is not the case for cocoa. OTA occurrence in cocoa, cocoa powder and cocoa marketed products has been reported in different countries (Tafuri et al., 2004; Burdaspal and Legarda 2003; Amezqueta et al., 2005) but there is scanty information on OTA contamination of cocoa products from Ghana. The present study investigated the contribution of pod integrity, pod
_____________________________________________________________ Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org
surface disease and sterility and effective drying to OTA contamination in cocoa. 2. Materials and Methods 2.1 Cocoa beans for the study The study was in two parts; the first part involved analysis of the level of OTA in cocoa beans, which had been obtained from harvested pods and processed on two cocoa plantations of the Cocoa Research Institute of Ghana located at Akim Tafo in the Eastern Region and Assin-Worakese in the Central Region of Ghana. The second part involved analysis of the level of OTA in already dried cocoa beans obtained from Wantram in the Western Region of Ghana, a subsidiary of the Cocoa Research Institute of Ghana. 2.2 Harvesting and Treatment of Pods At the farms where beans were obtained from harvested pods (Akim Tafo and Assin-Worakese), the cocoa pods were harvested using sickle and machete by staff of Cocoa Research Institute of Ghana during the 2009/2010 season. The Ripped pods were harvested over a period of 5 days and sorted into four categories; healthy pods, damaged pods, diseased and damaged pods and diseases pods. Healthy pods obtained at Akim Tafo were divided into three groups taken through heap, box and tray fermentation whereas those at Assin-Worakese were divided into two groups and fermented using heap and box fermentation. Diseased and damaged pods were fermented by heap fermentation. 2.3 Fermentation methods (i) Box fermentation: The wet beans were placed in wooden boxes one cubic meter in dimension (1m×1m×1m) and covered with plantain leaves and fermented for a period of 6 days during which they were aerated by turning them after every 2 days. (ii) Heap fermentation: The wet beans were tipped onto banana leaves placed on sticks on the ground. The heaps were then covered with other banana leaves. Heaps of about 50 kg each were conducted for beans from each pod category, i.e. healthy pods, broken/damaged pods, diseased pods and diseased and damaged pods. Fermentation lasted for a period of 6 days and beans were turned on every 2 days interval. (iii) Tray fermentation: In this case the wet beans were placed in wooden trays and surface covered with banana leaves. This fermentation lasted for 5 days as it has been experimentally confirmed that fermentation is faster when done in trays as compared to boxes and heaps.
Studies on factors which predispose fermented cocoa (Theobroma cacao) beans to mycotoxin contamination.
2.4 Surface sterilization
176
96 and 216 h into the start of drying.. All the collected beans were stored in deep freezers and later transported to the Department of Biochemistry, Cell and Molecular Biology, University of Ghana laboratory for OTA analyses in sealed bags. For the simulated protracted drying, samples were collected on the 1st, 2nd, 3rd and 4th week into start of drying in the sun. OTA was extracted from the cocoa beans by methods described by Bragulat et al. (2001) and Helica Biosystem respectively.
To investigate the effect of surface sterilization on incidence of OTA, some of the healthy and diseases pods harvested at Akim Tafo were divided into two groups; first group of pods were surface sterilized by 30% sodium hypochlorite and broken, beans scooped out and the second group of pods was broken open without surface sterilization. The beans were fermented using heap fermentation. 2.5 Drying
Fifty grams of the beans (whole beans) were ground until 50% of the resulting powder could pass through a 20 mm mesh screen. Ten grams of the resulting powder was weighed into a container and 50 ml of 70% methanol added. This mixture was mixed vigorously for 5 min and then centrifuged at 10,000 g for 5 min to clarity. About 100 µl of clear extract was taken and diluted with 900 µl of 70% methanol. The total solution of 1 ml was used for OTA quantification.
Beans from all the fermentation regimes were air dried on a platform made of raised raffia for a period of 9 days during which the beans were turned daily to allow for proper air circulation, effective drying and also to pick out rotten or damaged beans. A controlled category of dried beans was obtained in which improper drying due to reduced sunshine as it is the case during rainy season was simulated. This was done by drying beans in the open sun for short durations of (2 h) each day for a period of 4 weeks.
OTA levels were quantified using commercially available kit (96-well kit purchased from Helica Biosystem Inc, USA). All reagents were brought to room temperature before use. Equal numbers of mixing wells and antibody coated wells were placed in microwell holders. Exactly 200 µl of assay diluents were dispensed into each mixing well, 100 µl of each standard and prepared sample was added to the appropriate mixing wells containing the assay diluents.
2.6 Extraction and Determination of OTA levels One kilogram of dried beans was collected at intervals of 48 h,
Table 1 Levels of OTA recorded during drying of fermented cocoa beans at Akim Tafo, Treatment
Sampling time (hours after start of drying)
Healthy pods (heap fermented)
Healthy pods (box fermented)
Diseased pods (heap fermented)
Damaged/broken pods (heap fermented)
Healthy pods (tray fermented)
Diseased and fermented)
damaged
pods
(heap
OTA concentration (µg/kg)
96
0.00 ± 0.00 b, c, d, f, e
216
0.00 ± 0.00 b, c, d, e, f
96
0.03 ± 0.00 e, b, a
216
0.03 ± 0.00 f, d, a, e
96
0.02 ± 0.00 e, b, a
216
0.11 ± 0.00 e, b, c, a
96
0.05 ± 0.00 f, d, c, a
216
0.18 ± 0.00 f, d, b, c, a
96
0.05± 0.00 f, d, c, a
216
0.04 ± 0.00 f, d, e, a
96
0.02 ± 0.00 e, b, a
216
0.10 ± 0.00 e, b, c, a
Values are mean ± standard error of mean for three different measurements. The exponents (a, b, c, d, e, and f) represent significant difference between beans sampled on 96 h, whereas (a, b, c, d, e and f) represent significant difference between beans sampled on 216 h respectively. (a ,a) – Healthy pods heap fermented, (b, b) – healthy pods tray fermented, (c, c) – healthy pods box fermented, (d, d) – diseased and damaged pods heap fermented, (e, e) – damaged/broken pods heap fermented and (f, f) – diseased pods heap fermented. An OTA concentrations followed by a letter (superscript) indicates significant difference between it and the identity of the superscript at (P
