Filamentous fungi producing ochratoxin a during cocoa processing in Cameroon.

Abstract

Ochratoxin A (OTA) is the main mycotoxin occurring in cocoa. A study was conducted in Cameroon to assess how filamentous fungi and toxigenesis were affected by the type of cocoa post-harvest treatment (boxes or heaps). The filamentous fungi isolated were almost identical when fermentation was carried out in boxes or heaps, with the presence of abundant black Aspergillus filamentous fungi: A. niger and A. carbonarius. Filamentous fungi were more abundant at the end of the harvesting season. Factors affecting bean integrity (poor handling, deferred processing) resulted in a qualitative and quantitative increase in contamination, when the total number of filamentous fungi could reach a maximum value of 5.5+/-1.4x10(7) CFU g(-1) and black Aspergilli a maximum value of 1.42+/-2.2x10(7) CFU g(-1). A toxigenesis study showed that Aspergillus carbonarius was the main OTA-producing strain isolated. Its maximum production could reach 2.77 microg g(-1) on rice medium. Aspergillus niger strains did not always produce OTA and their toxigenesis was much lower. Fermented dried cocoa from poor quality pods was the most contaminated by OTA: up to 48 ng g(-1).

Full text

Page 1

Short communication
Filamentous fungi producing ochratoxin a during
cocoa processing in Cameroon
Pauline Mounjouenpoua, Dominique Gueuleb, Angélique Fontana-Tachonc, Bernard Guyotb,
Pierre Roger Tondjea, Joseph-Pierre Guiraudc,⁎
aInstitut de Recherche Agricole pour le Développement, BP 2067, Yaoundé, Cameroon
bUMR Qualisud, CIRAD, TA B-95/16, 73 Av. JF Breton, 34398 Montpellier Cedex 5, France
cUMR Qualisud, cc023, Université Montpellier 2, Place E. Bataillon, 34095 Montpellier Cedex 5, France
Received 7 March 2007; received in revised form 15 October 2007; accepted 6 November 2007
Abstract
Ochratoxin A (OTA) is the main mycotoxin occurring in cocoa. A study was conducted in Cameroon to assess how filamentous fungi and
toxigenesis were affected by the type of cocoa post-harvest treatment (boxes or heaps). The filamentous fungi isolated were almost identical when
fermentation was carried out in boxes or heaps, with the presence of abundant black Aspergillus filamentous fungi: A. niger and A. carbonarius.
Filamentous fungi were more abundant at the end of the harvesting season. Factors affecting bean integrity (poor handling, deferred processing)
resulted in a qualitative and quantitative increase in contamination, when the total number of filamentous fungi could reach a maximum value of
5.5±1.4×107CFU g−1and black Aspergilli a maximum value of 1.42±2.2×107CFU g−1. A toxigenesis study showed that Aspergillus
carbonarius was the main OTA-producing strain isolated. Its maximum production could reach 2.77 μg g−1on rice medium. Aspergillus niger
strains did not always produce OTA and their toxigenesis was much lower. Fermented dried cocoa from poor quality pods was the most
contaminated by OTA: up to 48 ng g−1.
© 2007 Elsevier B.V. All rights reserved.
Keywords: OTA; Cocoa; Filamentous fungi; Aspergillus carbonarius; Aspergillus niger
1. Introduction
Ochratoxin A (OTA) is a toxic secondary metabolite
produced by several species of Aspergillus and Penicillium
genera. It attracts particular attention through the damage it does
to the organism of humans and animals (Abarca et al., 1998). It
has nephrotoxic (Mantle and McHugh, 1993), immunotoxic,
teratogenic and carcinogenic properties (Kuiper-Goodman and
Scott, 1989; Kuiper-Goodman, 1996; Höhler, 1998). OTA has
been associated with Balkan Endemic Nephropathy (BEN) and
tumour development in the urinary tract (Mantle and McHugh,
1993). Following experiments on animals, the International
Agency for Research on Cancer (IARC, 1993) classed OTA as
carcinogenic for humans (group 2B).
Ochratoxin A is mainly produced byAspergillus carbonarius,
A. niger and A. ochraceus in tropical zones, and by Penicillium
verrucosum and P. nordicum in temperate zones (Pitt et al.,2000;
Abrunhosa, et al., 2001; O'Callaghan et al., 2003). Studies have
beenconductedtodetermine the degreeofOTAcontaminationin
several foodstuffs and drinks (Thirumala-Devi et al., 2001; Pittet
etal.,1996;WHO,2001;GareisandScheuer,2000;Skaug,1999;
Blanc et al., 1998; Hurst and Martin, 1998; Jorgensen, 1998).
Given its existence in several consumer products, consumer
exposure to OTA is increasing. In order to protect consumers, the
European Union has drawn up a standard defining tolerable
contamination limits. Other products, such as cereals, coffee and
wine are already covered by international regulation (European
Available online at www.sciencedirect.com
International Journal of Food Microbiology 121 (2008) 234–241
www.elsevier.com/locate/ijfoodmicro
⁎Corresponding author.
E-mail address: guiraud@univ-montp2.fr (J.-P. Guiraud).
0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2007.11.017

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Commission, 1995; Règlement (CE) n°472/2002 ), which is not
yet the case for cocoa. Given the extent of cocoa consumption
worldwide, the European Union will not delay in defining
maximum contamination limits.
World cocoa production is estimated at 3592000 tons (ICCO,
2006). Fermentation is the main stage in cocoa post-harvest
processing. It is generally carried out in a traditional manner by
spontaneous fermentation. First of all, there is colonization by
yeasts, followed by lactic bacteria, and then by acetic bacteria,
which are finally replaced by aerobic sporulated bacilli (Schawn
and Wheals, 2004; Thompson et al., 2001; Lopez and Dimick,
1995; Lehrian and Patterson, 1983). Recent studies dealed with
yeast (Jespersen et al., 2005) and bacteria (Camu et al., 2007)
populations associated with cocoa fermentations. Their succes-
sion and respective implication during fermentation were
investigated using molecular-based methods and the under-
standing of the process was subsequently improved (Nielsen
et al., 2007). However, very few studies exist on cocoa
filamentous fungi during technological treatments. The main
speciesisolatedduringnaturalfermentationofcocoainIndonesia
comprise Penicillium citrinum, Kloeckera apis, Saccharomyces
cerevisae, Candida tropicalis, Lactobacillus cellobiosus, Lacto-
bacillus plantarum and Acetobacter pasteurianus (Ardhana and
Fleet, 2003). In the Dominican Republic, a predominance of
yeasts of the genera Kloeckera and Candida is found at the
beginning of fermentation, followed by Lactobacillus pentosus,
Lactobacillus paracasei subsp. paracasei and Lactobacillus
brevis as the lactic bacteria and Acetobacter lovaniensis as the
main acetic bacterium (Lagunes-Gálvez et al., 2007). Whilst
several studies have been carried out on the evolution of
filamentous fungi in coffee and its relation with OTA content
during post-harvest processing (Suàrez-Quiroz et al., 2004;
Wilkens and Jörissen, 1999; Studer-Rohr et al., 1995; Micco
et al., 1989), that is not the case for cocoa. A microbiological
analysis on cocoa samples from 9 producing countries led to the
isolation of Aspergillus fumigatus and Rhizomucor pusillus
(Niles, 1981). There have been no studies on filamentous fungi
and OTA-producing species in cocoa depending on the type of
post-harvest processing.
High contamination frequencies have been found in cocoa
samples and by-products. Burdaspal and et Legarda (2003)
showed that OTA was found in 99.7 % of chocolate and cocoa
powder samples. Contamination of 81.3% was also described in
cocoa by-products by Miraglia and Brera (2002). Tafuri et al.
(2004) found OTA contamination of between 0.22 and 0.77 μg
kg−1in 10 samples of cocoa powder found on the Italian
market. A study involving 46 cocoa samples of different origins
found that 63 % of samples were contaminated by OTA, with an
average contamination of 1.71 μg kg−1(Amézqueta et al.,
2004). A maximum content of 100 μg kg−1was obtained with
cocoa contaminated artificially (Hurst and Martin, 1998).
Shelling by hand helped to reduce contamination levels in
cocoa beans by more thane 95% (Amézqueta et al., 2005).
The purpose of our study was to list and identify the fungi
that colonize cocoa beans at different stages of processing,
depending on the type of post-harvest process, and to study their
potential for producing OTA.
2. Materials and methods
2.1. Cocoa
Thecocoapods(TheobromacacaoL.)wereharvestedbyhand
during the 2005 cocoa season in the Kumba region of Cameroon.
2.2. Cocoa fermentation
Two types of fermentation were studied: box fermentation,
where the beans were placed in wooden boxes measuring
45 cm×45 cm×45 cm, and heap fermentation where the beans
were tipped onto banana leaves placed on the ground. Fermenta-
tion was carried out in each case using 50 kg of beans. The heap
wasthencoveredwithotherbananaleaves.Afterharvestinginthe
field, the pods were opened either immediately or later. De-
pendingonthetypeoffermentation,podconditionordelayinpod
opening, four treatment variants were investigated (Fig. 1): heap
fermentation of beans from whole pods that had been opened
immediately (T1), box fermentation of beans of the same type
(T2), heap fermentation of beans from whole pods opened after
10days(T3)heapfermentationofbeans fromdamagedpodsalso
openedafter 10days(T4).Inthe lastcase,the podswerepartially
opened after harvesting. Natural drying (in the sun) was carried
out for between 5 and 10 days. Three fermentations of each type
were performed during the cocoa season: at the beginning
(September–October),inthemiddle(October–November)andat
the end of the season (November–December).
2.3. Sampling
Cocoa samples were taken at different stages of processing
(Fig. 1). They involved unfermented beans (A), fermented
Fig. 1. Cocoa post-harvest processing (⁎sampling). T1: Heap fermentation of
beans from whole pods with immediate pod opening. T2: Box fermentation of
beans from whole pods with immediate pod opening. T3: Heap fermentation of
beans from whole pods with pod opening deferred by 10 days. T4: Heap
fermentation of beans from wounded pods with pod opening deferred by
10 days. A, B, C: Sampling stages.
235P. Mounjouenpou et al. / International Journal of Food Microbiology 121 (2008) 234–241

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undried beans (B), and fermented sun-dried beans (C). In each
case, 300 g cocoa samples were taken for microbiological
analyses. OTA quantification was performed on dry beans (dry
matter N90%). The samples taken at stages A and B were dried
for 48 h in the oven at 30 °C to reproduce natural drying,
bypassing any contamination occurring at that stage.
2.4. Microbiological analyses
The filamentous fungi population was enumerated by
inoculation on the surface of PDA medium (Biokar Diagnostics,
Beauvais, France). The inoculum was obtained by soaking 15
cocoa beans in 90 mL of a peptone water solution (0.1% w/v;
Biorad, Marnes la Coquette, France) for 10 min (Hocking and
Pitt, 1980). The result was expressed in CFU g−1for the total
filamentous fungi and for “black Aspergillus”. In order to de-
termine the infection percentage and increase numbering sensitiv-
ity, direct plating was carried out at the same time, aseptically
placing cocoa beans (3 beans per dish) on the surface of dishes
containing PDA medium (Hocking and Pitt, 1980). The dishes
wereincubatedat25°Cfor5to7daysandtheresultwasexpressed
as thepercentageof infectedbeans.Thefilamentous fungi isolates
wereselectedrandomlyaccordingtophenotypiccriteria(3isolates
for each phenotypic group). Isolates were identified according to
morphological criteria (Samson et al., 1995).The identification of
Aspergillus and Penicillium filamentous fungi was confirmed
using molecular techniques by the Fungi and Yeasts Culture
Collection at the Catholic University of Leuven in Belgium
(BCCM™/MULC Culture Collection). For each sample, the
frequency of A. carbonarius and Aspergillus niger isolates was
estimated in relation to total filamentous fungi.
2.5. Study of OTA production
Foreachstrainisolated,asuspension(3×106ofconidiamL−1)
wasmadeupbyscrapingaPDAculturedishwithasalinesolution
containing 0.01% Tween® 80 (P1754, Sigma-Aldrich, L'Isle
d'Abeau,France).FivemLofthesuspensionweredepositedinthe
centre of a dish of PDA medium which was incubated at 25 °C.
After 20 days incubation, direct extraction was carried out from 3
agar discs taken from the centre of the colony. Extraction was
carried out in 2.5 mL of solvent (methanol/formic acid 25:1 v/v)
for 15 min in an ultrasound bath.
In order to test production on cocoa, 50 g of cocoa beans
(verifiedOTA-free)wereinoculated with8 mLofa suspension of
50×106conidia mL−1and incubated at 25 °C for 20 days.
Extraction was carried out in an acetonitrile/water solution
(60:40v/v)for40min.Solvents were fromSigma-Aldrich(L'Isle
d'Abeau,France)(HPLCgrade).OTAproductionwasalsosought
on rice using the FDA method (Tournas et al., 2001).
In all cases, OTA was quantified on extracts by HPLC with
fluorimetric detection (Shimadzu LC-10 ADVP, Japan) (Naka-
jima et al., 1997). The operating conditions were as follows:
100 μL injection loop, C18 reverse phase HPLC column, ODS
5 μm (Supelco, Interchim, Montluçon, France) with an identical
pre-columnthermostaticallycontrolledat35°C,anisocraticflow
of1mL/min,anexcitationwavelengthof333nmandanemission
wavelength of 460 nm. Contents were calculated from a cali-
brationcurveestablishedfromastandard(1μgmL−1;refPD226
R. Biopharm Rhône Ltd, Glasgow, UK).
2.6. OTA quantification in cocoa beans
The dried cocoa bean samples were frozen at −80 °C, then
ground.Fifty grams ofgroundbeans wereextracted in200mLof
solvent(acetonitrile/water,60/40,v/v).FourmLoffilteredextract
were diluted in 44 mL of phosphate buffer. The mixture was
purified on an immunoaffinity column (Ochraprep, Rhône
Diagnostics, Scotland). OTA was eluted by 3 mL of methanol
and evaporated till dry in a nitrogen stream at 70 °C. The residue
was resuspended in 1 mL of the mobile phase (water/acetonitrile/
acetic acid, 51:48:1 v/v). Quantification was by HPLC using the
previously described method.
3. Results
3.1. Changes in filamentous fungi species during cocoa
processing
Table 1 gives the identification of the total filamentous fungi
in cocoa samples from the different fermentation operations.
They mostly belonged to the genera Penicillium, Aspergillus,
Mucor, Geotrichum, Trichoderma, Rhizopus, and Fusarium, with
some species known to produce OTA (A. niger, A. carbonarius).
There was no significant difference for the isolated filamentous
Table 1
Identification of the filamentous fungi isolated during technological treatments
Treatment Sampling
stage
Filamentous fungi isolated
T1A
B
–
A. versicolor, Mucor spp, A. niger, Geotrichum spp,
A. fumigatus, Fusarium spp, Rhizopus nigricans
A. tamarii, A. fumigatus, Rhizopus nigricans, A. niger
–
A. versicolor, A. fumigatus, A. tamarii, Rhizopus
nigricans, Fusarium spp, A. niger
Rhizopus nigricans, A. fumigatus, A. tamarii, A. niger
P. crustosum, Fusarium spp, A. tamarii,
P. sclerotiorum, A. flavus, A. fumigatus, Mucor spp,
Rhizopus nigricans, A. niger
Rhizopus nigricans, Mucor spp, A. fumigatus,
Syncephalastrum racemosum, A. tamarii,
P. sclerotiorum, A. flavus, Geotrichum spp,
Trichoderma spp, A. niger
Geotrichum spp, Mucor spp, A. fumigatus, A. flavus,
A. tamarii, A. niger
Scopulariopsis spp, A. niger, Syncephalastrum
racemosum, A. fumigatus, Rhizopus nigricans, Mucor
spp, P. crustosum, A. carbonarius
A. versicolor, Rhizopus nigricans, Mucor spp,
Scopulariopsis spp, Syncephalastrum racemosum,
A. niger, P. crustosum
P. crustosum, P. sclerotiorum, Fusarium spp,
Scopulariopsis spp, Rhizopus nigricans, A. flavus,
Trichoderma viride, A. niger, A. carbonarius
C
A
B
T2
C
A T3
B
C
T4A
B
C
A, B, C, T1–T4 are defined on Fig. 1.
236 P. Mounjouenpou et al. / International Journal of Food Microbiology 121 (2008) 234–241

Page 4

fungi depending on fermentation type(heap or box). Pod integrity
and, to a lesser degree, a delay in pod opening affected the
qualitative diversity of the filamentous fungi. Wounded pods
revealed high A. carbonarius, A. niger and Fusarium spp
proliferation in the pod openings.
Table 2 gives the quantification of total filamentous fungi
and black filamentous fungi isolated during the technological
treatments. Filamentous fungi were found for all the conditions,
but they varied in number depending on the sampling stage.
Stage B had the highest contamination rate for both total fila-
mentous fungi and for black Aspergillus. Maximum concentra-
tions of 5.5±1.4×107CFU g−1and 1.4±0.2×107CFU g−1
were obtained for those two categories respectively. It should be
noted that the number of black Aspergillus filamentous fungi
(A. carbonarius and A. niger) was not identical throughout the
cocoa harvest season. Those Aspergillus were less frequent at
the beginning of the season, and were only encountered at stage
B(afterfermentation),exceptinwoundedpods.Cocoasfromthe
middle and end of the season were more contaminated, irre-
spective of the sampling stage, and that contamination increased
in line with pod damage. Identification of the isolated strains
revealed that A. niger was the predominant strain (90 to 100% of
black Aspergillus). A. carbonarius was mostly found in un-
fermented beans (A) from wounded pods.
Direct plating confirmed high cocoa bean contamination by
filamentous fungi (Table 3). The contamination rates by total
filamentous fungi was 100% irrespective of treatment type.
Black filamentous fungi were found after fermentation and
drying(whenpodswereopenedimmediately)andatallstagesof
processing when pod opening was delayed, and with wounded
pods. These results confirmed the counting results.
3.2. OTA production by filamentous fungi
The ability of strains of the main species of filamentous fungi
isolated (A. fumigatus, A. tamarii, A. versicolor, A. carbonarius,
A. niger, Penicillium sclerotiorum, P. paneum, P. crustosum) to
produce OTAwas first tested for a large number of strains (310)
on PDA medium. Production was then quantified on rice
medium (Tournas et al., 2001) and on cocoa medium for the
Table 2
Quantification of total and black Aspergilli isolated during technological treatments
Sampling stage Total filamentous fungi (CFU g−1) Black Aspergilli (CFU g−1) A. carbonarius (% of black Aspergilli)
Beginning of season
(September–October)
T1A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
nd
4.0±1.4×107
3.7±0.5×106
nd
4.5±0.8×107
4.0±1.9×104
2.6±0.7×105
5.0±0.8×107
3.5±2.6×104
1.6±0.6×105
5.5±1.4×107
1.0±0.4×106
nd
7.7±1.9×106
5.2±0.7×106
nd
2.3±0.5×106
4.2±2.0×104
3.2±0.4×106
2.0±0.3×107
4.9±0.7×105
6.0±1.7×106
2.0±0.3×107
7.8±0.8×106
nd
7.7±1.9×106
5.2±0.7×106
nd
2.3±0.5x 106
4.2±2.0×104
1.8±0.9×105
5.4±0.5×107
3.1±0.5×106
2.9±0.4×107
2.5±0.3×107
2.5±0.5×106
nd
5.2±1.8×106
nd
nd
1.3±0.2×106
nd
nd
8.7±4.2×105
nd
1.4±0.5×105
2.2±0.2×106
1.3±0.57×105
nd
2.5±0.6×106
3.5±0.5×106
nd
nd
2.8±1.3×104
5.5±0.7×105
3.4±0.5×106
4.9±0.7×106
1.6±0.5×106
1.4±0.2×107
4.1±0.5x 106
nd
1.5±0.2×106
2.0±0.2×105
nd
3.0±08.×106
4.6±0.7×104
9.0±4.0×103
2.7±0.3×106
2.5±0.4×105
7.0±0.9×106
4.0±0.5×106
3.0±0.6×105
–
0
–
–
0
–
–
–
0
75
0
0
–
0
0
–
–
0
0
0
0
38
0
0
–
0
0
–
0
0
0
0
0
50
0
0
T2
T3
T4
Mid-season
(October–November)
T1
T2
T3
T4
End of season
(November–December)
T1
T2
T3
T4
A, B, C, T1–T4 are defined on Fig. 1.
nd: not detectable (quantification limit 10 CFU g−1).
237P. Mounjouenpou et al. / International Journal of Food Microbiology 121 (2008) 234–241

Page 5

most toxigenic strains (Table 4). No OTA production was found
with A. fumigatus, A. tamarii, A. versicolor, P. sclerotiorum,
P. paneum, P. crustosum. Of the fifty-three A. carbonarius
isolates, only two were studied in detail. Irrespective of the
culture medium (rice or cocoa), those isolates revealed strong
toxigenesis which varied depending on the culture medium.
OTA production was greater on rice medium with a content of
573.4 to 2.772 ng g−1after twenty days' culture. On cocoa
medium, values of 39.2 to 84.5 ng g−1after 8 days' culture and
50.6 to 110.7 ng g−1after 20 days' culture were obtained. Of the
Table 4
OTA production by identified filamentous fungi
OTA production (ng g−1)
Isolate
Rice medium
after 20 days
Cocoa medium
8 days
Cocoa medium
after 20 days
A. carbonarius 1
A. carbonarius 2
A. niger 1
A. niger 2
A. niger 3
573.4
2772
nd
3.5
3.6
39.2
84.5
0.5
0.1
nd
50.6
110.7
nd
0.2
0.05
A, B, C, T1–T4 are defined on Fig. 1.
nd: not detectable (detection limit 0.03 ng g−1).
A. carbonarius 1: Beginning of season, treatment T4, sampling stage A.
A. carbonarius 2: Mid-season, treatment T3, sampling stage C.
A. niger 1: Beginning of season, treatment T4, sampling stage A.
A. niger 2: Beginning of season, treatment T2, sampling stage B.
A. niger 3: End of season, treatment T4, sampling stage B.
Table 3
Variation in contamination rate by total and black filamentous fungi during
cocoa processing (Infected beans/total beans)
Treatment Sampling
stage
Rate of
contamination by
filamentous fungi
Rate of
contamination
by black
Aspergilli
Beginning of
season
(September–
October)
T1A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
0/6
6/6
6/6
0/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
0/6
6/6
6/6
0/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
0/6
6/6
6/6
0/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
6/6
0/6
0/6
2/6
0/6
5/6
2/6
0/6
0/6
0/6
4/6
3/6
3/6
0/6
2/6
5/6
0/6
0/6
2/6
2/6
3/6
5/6
4/6
3/6
5/6
0/6
0/6
2/6
0/6
0/6
2/6
1/6
4/6
4/6
2/6
3/6
5/6
T2
T3
T4
Mid-season
(October–
November)
T1
T2
T3
T4
End of season
(November–
December)
T1
T2
T3
T4
A, B, C, T1–T4 are defined on Fig. 1.
Table 5
Correlation between OTA content in fermented dried cocoa, treatment type and
associated filamentous fungi
Treatment OTA in beans
(ng g−1)
Main associated
filamentous fungi
Beginning
of season
T1A
B
C
A
B
Nd
0.05±0.01
0.02±0.02
Nd
0.04±0.02
/
A. versicolor, A. nigera
A. tamarii
/
A. versicolor, A. nigera,
A. fumigatus, P. paneum
A. tamarii
P. sclerotiorum
A. versicolor
A. fumigatus, A. tamarii
A. carbonariusb, A. nigera
A. versicolor
A. nigera, P. sclerotiorum,
P. crustosum
/
A. nigera, P. paneum
A. nigera, P. paneum
/
A. tamarii, P. paneum
A. nigera, A. tamarii,
P. paneum
A. tamarii, A. nigera,
P. sclerotiorum
A. tamarii, A. nigera,
P. paneum
A. nigera
A. carbonariusb, A. nigera,
P. paneum
A. nigera, A. tamarii
A. nigera
/
A. nigera, P. paneum
A. nigera
/
A. nigera, A. tamarii,
P. paneum
P. paneum, A. nigera,
A. nigera, P. paneum
P. paneum
A. tamarii, A. nigera
A. carbonariusb, A. nigera,
P. crustosum
A. nigera, P. crustosum
A. nigera
T2
C
A
B
C
A
B
C
0.15±0.01
0.02±0.01
0.17±0.04
0.09±0.01
12.14±0.10
0.41±0.04
0.16±0.02
T3
T4
Mid-seasonT1 A
B
C
A
B
C
nd
0.13±0.02
0.03±0.00
nd
nd
0.01±0.00
T2
T3A0.03±0.00
B 0.25±0.20
C
A
0.05±0.00
22.20±0.91T4
B
C
A
B
C
A
B
1.14±0.07
1.01±0.02
nd
0.06±0.01
0.12±0.01
nd
0.27±0.01
End of
season
T1
T2
C
A
B
C
A
0.06±0.00
0.08±0.01
nd
0.06±0.03
48.01±0.75
T3
T4
B
C
4.18±0.24
5.40±0.41
A, B, C, T1–T4 are defined on Fig. 1.
aToxigenic.
bVery toxigenic.
238P. Mounjouenpou et al. / International Journal of Food Microbiology 121 (2008) 234–241

Page 6

145 A. niger isolates, only 3 were studied in detail. Compared to
A. carbonarius, the toxigenesis of the three A. niger strains was
much lower. On rice medium, OTA production was from 0.03 to
3.6 ng g−1as opposed to 0.03 to 0.2 ng g−1on cocoa medium
after 20 days' culture.
3.3. OTA quantification in fermented dried cocoa
Fermented dried cocoa from the different fermentation
operations was analysed to check for the existence of OTA.
Table 5 gives the OTA contents found, along with the toxigenic
flora associated with those cocoa beans.
Whatever the sampling stage, bean contamination by OTA
was low for box and heap fermentation when the beans came
from intact pods. In that case, OTA contents were between nd
(not detectable) and 0.27 ng g−1, which remained below 2 ng
g−1(limit defined for wine) and 5 ng g−1(limit defined for
roasted coffee). The toxigenic microflora associated with beans
contaminated by OTA mostly consisted of A. niger. When pods
were wounded, a maximum content of 48.02 ng g−1was
observed, which was well over tolerable rates. The toxigenic
species associated with those beans were A. carbonarius and
A. niger.
4. Discussion
Filamentousfungiholdanimportantpositionintheecologyof
cocoa beans during fermentation (Roelofsen, 1958; Schawn and
Wheals, 2004). The main filamentous fungi isolated during our
workwereA.fumigatus,A.tamarii,A.versicolor,A.carbonarius,
A. niger, P. sclerotiorum, P. paneum and P. crustosum, Mucor
spp, Rhizopus spp, Fusarium spp and Trichoderma spp. Our
results differed from those quoted in the literature for the
filamentous fungi associated with fermented beans (Marvalhas,
1966) or dried beans (Ciferri, 1931; Bunting, 1928; Dade, 1928).
In those publications, A. fumigatus, Aspergillus glaucus, Mucor
spp and Penicillium spp were isolated. Marvalhas (1966) isolated
P. citrinum from fermented beans. According to the literature,
filamentous fungi associated with fermentation and drying are
different. With the exception of A. fumigatus, and Mucor spp,
the other species found are not described as being associated
with cocoa.
Irrespective of pod condition and type of fermentation
(boxes or heaps), a large increase in filamentous fungi species
was found after fermentation (stage B). That might have been
explained by the existence of sweet mucilage, which is highly
conducive to filamentous fungi development. However, drying
helped to reduce the flora, as susceptible species disappeared to
the benefit of soil-born and more generally environmental flora
which led to contamination during solar drying. A. niger was
foundunderallconditions,fromfreshbeantofermentedanddried
bean. Contamination byA. carbonarius was particularlyfound in
unfermented beans from damaged pods with deferred pod
opening. That high contamination may have been due to the
fact that when pods were partially opened, the cocoa beans came
into direct contact with the air and ground. With fermentation,
there was competition between the different species present and
those with rapid growth (Mucor, Rhizopus spp) managed to
colonize first to the detriment of Aspergillus and Penicillium.
A study of OTA production by the isolated filamentous fungi
revealed that A. fumigatus, A. tamarii, A. versicolor,
P. sclerotiorum, P. paneum, and P. crustosum did not produce
OTA. Contrary to our results, the literature indicates that
A. fumigatus and A. versicolor can produce OTA in cereals
(Rizzo et al., 2002). P. sclerotiorum has also been reported to
produce OTA (Frisvad et al., 2004). It is likely that differences
exist between strains of different origin, with a substrate effect.
Our results showed that only A. niger and especially
A. carbonarius were able to produce OTA in cocoa. All the
A. carbonarius isolates produced OTA, whereas 70 % of
A. niger isolates were toxigenic but only with a low production
level (Table 4). This is an interesting unusual result considering
that Taniwaki et al. (2003) found that 75 % of Aspergillus
ochraceus strains and 3 % of A. niger isolates produced OTA in
coffee. In grapes, A. carbonarius were also the main OTA-
producing species: 97% of the A. carbonarius isolates and 3 %
of the A. niger aggregate isolates were OTA-positive (Lasram
et al., 2007). These results illustrated well the biodiversity of the
black Aspergilli according to their natural environment.
OTA production by isolated species varied depending on the
substrate. It was greater on rice medium than on cocoa medium
(Table 4). Our results tallied with those of other authors which
revealed the effect of the substrate on mycotoxin production
(Kokkonen et al., 2005).
AsshowningrapesbyBellietal.(2006),thehighOTAlevelin
cocoa beans produced byheap fermentationfromwounded pods,
withpodopeningdeferredby10days(T4),couldbecorrelatedto
the presenceofA.carbonarius.TreatmentT4,duringwhichmost
A. carbonarius strains were isolated, thus gave the most
contaminated cocoa.
However, the statistical significance of these results has to be
improved by studies during other cocoa seasons in areas under
different environmental conditions.
Nielsen et al. (2005) have successfully studied the yeast
population dynamics during Ghanaian cocoa fermentations
using denaturating gradient gel electrophoresis.Inthesameway,
molecular-based methods could be useful tools to study the black
Aspergilligenetic biodiversityinrelationwiththeirtoxinogenesis.
It is known (Esteban et al., 2006) that drying and storage
conditions play a major role in the presence of OTA. Our results
show that contamination prior to processing also greatly in-
fluences end-quality and that good pod condition and immediate
pod opening can partly reduce the risks.
Acknowledgements
Our thanks to the Cooperation and Cultural Action Service
(SCAC) at the French Embassy in Cameroon, to CIRAD and to
the REPARAC project for funding this work.
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