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Journal of Microbiology,
Biotechnology and Fagbohun et al. 2011 1 (2) 204-214
Food Sciences
204
REGULAR ARTICLE
FUNGI ASSOCIATED WITH SPOILAGE OF DRIED COCOA BEANS DURING
STORAGE IN EKTI STATE OF NIGERIA
Emmanuel Fagbohun*, I. Anibijuwon, Oluwole Egbebi and Opeyemi Lawal
Address*: Emmanuel Fagbohun, Ekiti State University, Ado Ekiti, Ekiti State. Faculty
Science, Department of Microbiology, Ado Ekiti, Ekiti, Ekiti State, Nigeria.
fagbohundayo@yahoo.com, opeyemil@ymail.com, 08035070548.
ABSTRACT
Fungi associated with cocoa beans during storage were surveyed in some stores in
Ado, Ise, Emure and Ikere in Ekiti State of Nigeria during July-December 2010. The following
fungi were consistently isolated from mouldy cocoa bean samples, namely; Aspergillus niger,
Aspergillus flavus, Botryodiplodia theobromae, Fusarium spp., Mucor spp., Neurospora spp.,
Penicillium spp., and Phytophthora palmivora. The various fungi were isolated using
washing, direct and dilution plate methods respectively. At Ado, the following fungi;
Aspergillus flavus, Aspergillus niger, Botryodiplodia theobromae, Mucor spp., Neurospora
spp. Penicillium spp., Phytophthora palmivora, Rhizopus spp. were occasionally isolated from
stores that were not properly ventilated. At Ise, Phytophthora palmivora, Mucor spp. and
Penicillium spp. were commonly isolated, while Aspergillus flavus, Aspergillus niger and
Rhizopus spp. were occasionally isolated in stores where the bags were kept on the bare floor.
At Emure, Aspergillus spp. and Phytophthora palmivora were commonly isolated, while
Aspergillus spp., Rhizopus spp., Neurospora spp., Botryodiplodia theobromae, Fusarium spp.
and Mucor spp., were occasionally isolated from stores with leaking roofs. At Ikere,
Aspergillus niger, Aspergillus flavus, Phytophthora palmivora, Fusarium spp., Mucor spp.
and Penicillium spp. were consistently isolated while Rhizopus spp., and Botryodiplodia
theobromae were occasionally isolated. Some of these fungi gain access to the beans during
fermentation, drying, storage and shipment to the foreign countries. Some of these isolated
JMBFS / Fagbohun et al. 2011 1 (2) 204-214
205
fungi have been reported by many workers to produce toxic substances which have serious
health implications in both man and animals.
Keywords: Survey, fungi, spoilage, storage, cocoa bean
INTRODUCTION
Cocoa belongs to the genus Theobroma in the family of the Sterculiaceae. But
recently, with the application of molecular marker, cacao was classified to belong to the family
Malvaceae (Alvenson et al., 1999). According to Opeke, (1992) Theobroma cacao is
cauliflorous and semi-deciduous and about 20 species of Theobroma are recognized.
Cocoa is a low-attitude crop. It grows from sea level up to an attitude of 700 metres. In
Nigeria and in other African countries where cocoa is grown a minimum of 25 percent of clay
is required for cocoa to do very well and healthy (Opeke, 1992; Aigbekaen, 2004; Sanusi
and Oluyole, 2005).
The cocoa pods attain maturity between 110 to 130 days depending on the variety,
from pollination to pod ripening (Opeke, 1992; Sanusi and Oluyole, 2005). To ensure the
production of quality beans, it is essential that only matured and ripe pods are harvested and
processed promptly (Hamzat, 2005). In West Africa there are two pods production seasons,
the main season – July to December and light crop season – January to April (Motamayor,
2002). Harvesting exercise is carried out regularly and frequently to prevent immature
ripening. For effective storage of excess cocoa pods, the use of modified packaging of cocoa
pods in transparent polythene films conserved the commercial qualities of cocoa beans, such as
cocoa butter, percentage moisture content, reduced growth and severity of decay (Aroyeun et
al., 2006).
Fermentation of cocoa beans is carried out in order to obtain a proper taste, colour;
flavour associated with cocoa products and also to kill the embryo to forestall germination.
There are several methods of fermentation. These include, heap fermentation, basket, sweat,
box and tray fermentation method. The most popular and frequently used is the tray method
(Hamzat, 2005; Aroyeun et al., 2006).
Stored cocoa is often damaged by insect, pests and most especially by moulds (Hamzat, 2004;
Aroyeun et al., 2006). This type of damage mostly results from the failure to dry beans
JMBFS / Fagbohun et al. 2011 1 (2) 204-214
206
properly. Production of good quality cocoa will therefore not only depend on proper
fermentation but also on correct drying methods. Various methods of drying the fermented
cocoa beans are used and these include sun drying and artificial drying. Most commonly used
method in Nigeria is sun drying and this depend on the climatic conditions (Motamayor,
2002).
The quality of commercial beans depends very largely on how well the fermentation has
been carried out (Hamzat, 2004). The period of fermentation of the cocoa bean determines
the quality of the product that will be produced by the bean. On the other hand,
microorganisms such as mould whose presence is detrimental to the quality of cocoa beans
gain access to them during fermentation (Hamzat et al., 2006).
ICCO (2004) reported the isolation of several fungi growing on fermenting cocoa
beans. Those of West Africa (Ghana) origin are Aspergillus fumigatus, Aspergillus tamarii
and Mucor pusillus. With increasing length of fermentation therefore cocoa beans have a
greater chance of being penetrated by mould which grows externally on them (Opoku et al.,
2007).
The extent to which internal mouldiness occur in stored cocoa bean by fungi is
inestimable. The cotyledons of the beans which is the mesh filament are affected and results
into colour change from cream to green, yellow, black, brown or white in the cocoa beans
(Opeke, 1992; ICCO, 2004).
These forms of structure are known as mould or microscopic fungi (Krasauskas et al.,
2006). Moreover, their presence in a bean could be readily seen with the naked eye especially
when they are advanced in growth. Mould increase the free fatty acid content of cocoa butter
and make referring of the fat essential before it can be used. In addition moulds do cause an
actual loss in weight of cocoa beans (Hamzat, 2004).
The aim and objective of this study was to isolate the fungi associated with the spoilage
of stored dried cocoa beans.
MATERIAL AND METHODS
Collection of Mouldy cocoa beans
Samples of mouldy cocoa beans were collected from early July to December 2010, in
some cocoa stores in Ise, Emure, Ado and Ikere. A total of 20 cocoa stores were visited
together in these towns.
JMBFS / Fagbohun et al. 2011 1 (2) 204-214
207
Fifty beans were picked randomly from cocoa bags from each store. A total of 1000
beans were picked. Immediately upon collections, all samples were further dried to stop
further mould growth (Lillehoj et al., 1975). The samples were kept in insect free bags,
labeled and transferred to the laboratory. The mouldy beans were separated from non affected
beans.
Isolation of Microorganisms from Mouldy Cocoa Beans
Direct Plating Method
From each bag 50 beans were examined randomly for internal mouldness. The beans
were washed twice with sterile distilled water by stirring. The cocoa beans were cut into two
halves along the longitudinal axis with a sterile dissecting knife. Using a sterile dissecting
forceps, mouldy cocoa beans were aseptically picked and placed on Malt Extract Agar and
incubated at 28oC for 7 days (Amusa, 2001 and Arotupin, 2004).
The fungi cultures were further subculture until pure colonies were obtained by successive
hypha tip transfers. The cultures were examined under a dissecting microscope to determine
the common fungi present.
Dilution Plate Method
This method was used to determine the type of fungi present in the mouldy cocoa
beans. However, 1g of each bean sample was grounded and 10ml of sterile distilled water was
added. This was shaken thoroughly and 1ml of the suspension was pipette into a sterile test
tube and mixed again. This was repeated until dilutions 10-1 to 10-5 was obtained and 1ml from
each of the dilution 10-3 to 10-5 was added to 18ml of molten Malt Extract agar. The plates
were swirled gently to obtain a thorough mixing. The plates were incubated at 28oC for 7days.
Fungal colonies were subculture until pure cultures were obtained (Atanda et al., 1990).
Washing Method
This method was carried out by weighing 1g of each sample into 10ml of sterile
distilled water in a sterile test tube. This was shaken thoroughly and serial dilution of 10-1 to
10-5 was prepared from the resulting solution. However, 0.1ml of dilution of 10-4 and 10-5 were
introduced into Malt Extract Agar plates. This was spread using a sterile glass spreader and
incubated at 28oC for 7 days.
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208
Identification of microorganisms
The isolates were examined under day light for the colour of the culture and further
examination was carried out using needle mount preparation method of Tuite (1961),
Crowley et al., (1969) and Burnett (1975).
Slide culture Method
The method of Crowley et al., (1969) and Dugan (2006) was used whereby a 1cm2 of
malt extract agar was cut from a plate approximately 2mm deep and placed on a sterile glass
slide. Fungus was maculated into the four vertical sides suing a sterile needle. A sterilized
cover slip was applied so that it over lapped the medium on all sides and the preparation was
placed on suitable support in a petri dish containing blotting paper soaked in 20% glycerol in
water. The preparation is kept moist at 26oC until adequate growth developed. After
removing the medium the fungus adherent to both cover slip and slide was examined. A drop
of alcohol added, followed by a drop of lactophenol blue and the preparation suitably covered
and examined under the low power objectives microscope.
RESULTS AND DISCUSSION
In this study a total of nine fungi were isolated and identified based on their cultural and
morphological characteristics. They are: Aspergillus niger, Aspergillus flavus, Botryodiplodia
theobromae, Fusarium spp., Mucor spp., Neurospora spp., Penicillium spp., and
Phytophthora palmivora.
The microorganisms isolated from mouldy cocoa beans from Ado, Ikere, Ise and Emure using
three major methods (Direct plating, Dilution method and Washing method) are shown on
Table 1.
This study showed that Aspergillus spp., Penicillum spp., Mucor spp., and
Phytophthora palmivora were isolated from all the locations using the three methods of
isolation, while those rarely isolated were Fusarium spp., Neurospora spp. and Botryodiplodia
theobromae. These results is in agreement with the work of ICCO (2004) who worked on
cocoa beans and reported the isolation of various species of fungi associated with internal
mouldiness of cocoa beans in Ibadan..
The results of this study also showed that Mucor spp., Rhizopus spp., Phythphora
palmivora and Aspergillus spp. were all isolated from cocoa beans from the three locations
JMBFS / Fagbohun et al. 2011 1 (2) 204-214
209
(Ado, Ise, Emure). This is in agreement with the report of ICCO (2006) who found that
Mucor spp., Rhizopus spp., Phythophthora palmivora and Aspergillus spp. were the principal
fungi causing spoilage of stored cocoa beans.
The isolation of Neurospora spp. was common to only Ado and Emure while
Botryodiplodia theobromae was common to Ado only. Isolation of Penicillium spp. and
Phytophthora palmivora was common to Ado, Ise, Emure and Ikare. Fusarium spp. was
common to Emure while Mucor spp. was common to Ikere. These results is in agreement with
the previous findings by Chatt (1953) who reported that these moulds gained asses to cocoa
bean at the various stages during the preparation of the crop from the market.
At Ikere, the following fungi were isolated using direct plate method namely, A. flavus,
P. palmivora, Penicillium spp., while A. niger, B. theobromae, Fusarium spp., and Mucor
spp. were isolated using dilution plate method and Rhizopus spp. was additionally isolated
using washing method. The isolated fungi are in agreement with the work of Oyeniran (1976)
who isolated most of the fungi from a survey of internal mouldiness of cocoa. These moulds
gained access when the pods have been damaged. Similarly Oyeniran (1976) recorded 66% of
internally mouldy cocoa beans which could have resulted from pre- harvest infection. Previous
findings by Broadbent et al., (1969) showed that brown and black cocoa beans contained
internally mould beans.
At Emure and Ise, the fungi isolated using direct plating and dilution plate method
respectively are A. niger, A.flavus, P. palmivora and Penicillium spp., while Rhizopus spp.
was isolated using washing method. These findings are in agreement with the work of
Oyeniran (1976) who isolated some of these fungi from a survey of mouldy cocoa beans. The
possible source of these moulds could have been during sun drying process or the handling
process during storage of the product. (ICCO (2004) reported an internal mouldiness of 25%
when drying was prolonged for 13-14 days as a result of dull weather. Copetti et al., (2011)
and Magalhães et al., (2011) also reported the isolation .Aspergillus flavus, Aspergillus niger,
Asperillus carbonarius, Aspergillus nomius, Aspergillus ochraceus and Penicillium paneum
Absidia corymbifera from the dried and stored cocoa beans.
In Ado, Aspergillus spp., B. theobromae P. palmivora, Mucor spp. were consistently
isolated using direct plating and washing methods while Neurospora spp. was isolated using
dilution plate method. The presence of these storage fungi are in agreement with the findings
of Mounjouenpou et al., (2007) and Sánchez-Hervás et al., (2008) who found A. niger,
A.carbonarius to be associated with the processing and storage of cocoa beans in Cameroon.
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Some of these fungi according to Fandohan et al., (2003) gain asses to cocoa beans during
storage either by air or by contamination from handlers of the stored cocoa beans.
The control of mouldiness of cocoa beans during drying is difficult. The wet season
prolong the drying process of cocoa bean thereby encouraging the growth of spoilage fungi.
Moreover, during fermentation fungi gain asses to bean when temperatures are extremely high.
A few species such as Aspergillus spp., and Mucor spp., has been frequently isolated
(Hamzat, 2005; Sanusi and Oluyole, 2005). Other factors that enhance penetration of beans
during this stage are germination and mechanical damage through which storage fungi gain
asses to the inside of the beans of the stored sundried cocoa.
Tab 1 A summary of fungi isolated from the cocoa stores during the survey using various
methods of isolation.
ISOLATED FUNGI
TOWNS AND METHOD
ADO
A, B, C
ISE
A, B, C
EMURE
A, B, C
IKERE
A, B, C
Aspergillus flavus + + + + - + + + - + + -
Aspergillus niger + - + + - + + + + - + +
Botryodiplodia theobromae + + - - - - + - - - + -
Fusarium spp. - - - - - - + - + - + +
Mucor spp. + - + + + - + - - - + +
Neurospora spp. - + + - - - + - + + - +
Penicillium spp. + + + + + + - + + + - +
Phytophthora palmivora + + + + + + + + + + + +
Rhizopus spp. - - - - - + - + - - - +
Legend:
Key Method of isolation
+ Present (A) = Direct plating method
- Absent (B) = Dilution plating method
(C) = Washing method
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CONCLUSION
Cocoa beans are of great economic importance and in order to maintain the quality,
they should be stored under controlled environment that would not be favourable for the
growth of fungal flora thereby preventing deterioration of the stored cocoa bean and reduction
in the chemical composition. This present study has revealed the various fungi associated with
stored cocoa bean at different towns in Ekiti State. However, apart from good hygiene, proper
handling and processing practice should be employed to reduce the contamination of stored
cocoa bean. The isolated fungi can degrade the cocoa bean as substrate thereby reducing the
market value, also making consumers especially the immunocompromised individual vulnerable
to microbial infection.
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