International Journal of Food Microbiology 148 (2011) 141–144

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International Journal of Food Microbiology

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Aflatoxigenic fungi and aflatoxin in cocoa

Marina V. Copetti a,⁎, Beatriz T. Iamanaka b, José Luís Pereira c, Maria H. Fungaro d, Marta H. Taniwaki b
a
Universidade Federal de Santa Maria (UFSM), Departamento de Tecnologia e Ciência de Alimentos, CEP 97105, 900, Santa Maria/RS, Brazil
b
Instituto de Tecnologia de Alimentos (ITAL), C.P. 139, CEP 13070-178, Campinas/SP, Brazil
c
Universidade Estadual de Campinas (UNICAMP), Departamento de Ciência de Alimentos, C.P. 6121, CEP 13083-862, Campinas/SP, Brazil
d
Universidade Estadual de Londrina (UEL), Centro de Ciências Biológicas, C.P. 6001, CEP 86051-990, Londrina/PR, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: This paper reports the occurrence of aflatoxigenic fungi and the presence of aflatoxins in 226 cocoa samples
Received 3 March 2011 collected on Brazilian farms. The samples were taken at various stages of fermentation, drying and storage.
Received in revised form 4 May 2011 A total of 819 potentially aflatoxigenic fungi were isolated using Dichloran 18% Glycerol agar after surface
Accepted 23 May 2011
disinfection, and identified by standard techniques. The ability of the fungi to produce aflatoxins was
Available online 27 May 2011
determined using the agar plug technique and TLC. The presence of aflatoxins in cocoa samples was
Keywords:
determined by HPLC using post-column derivatization with bromide after immunoaffinity column clean up.
Aflatoxin The aflatoxigenic fungi isolated were Aspergillus flavus, A. parasiticus and A. nomius. A considerable increase in
HPLC numbers of these species was observed during drying and storage. In spite of the high prevalence of
Aflatoxigenic fungi aflatoxigenic fungi, only low levels of aflatoxin were found in the cocoa samples, suggesting the existence of
Theobroma cacao limiting factors to the accumulation of aflatoxins in the beans.
Aspergillus flavus © 2011 Elsevier B.V. All rights reserved.
Aspergillus parasiticus
Chocolate

1. Introduction representative aflatoxin producer species naturally occurring in

agricultural commodities (Cary and Ehrlich, 2006). A. nomius, which
Aflatoxins represent the group of the most studied mycotoxins, is also reported as a major producer of aflatoxins, has a more restricted
especially due to their widespread occurrence in foods and toxico- occurrence (Kurtzman et al., 1987).
logical and carcinogenic potential associated with their consumption. The occurrence of aflatoxigenic species is high in tropical
Since aflatoxins have hepatotoxic, teratogenic, mutagenic and countries. Although a wide variety of foods are susceptible to aflatoxin
carcinogenic properties, many countries have imposed regulations contamination, they have been most commonly associated with
aiming to minimize the human exposure to aflatoxins (IARC, 1993). peanuts, pistachio, dried fruits, nuts, spices, figs, vegetable oils, cocoa
This legal restriction has resulted in rejection of products causing beans, corn, rice and cotton seed (JECFA, 1998; ROC, 2003).
great economic losses for producers, processors and marketers of Cocoa beans, the principal raw material of chocolate, have an
contaminated products (Cotty and Jaime-Garcia, 2007). astringent, unpleasant taste and flavor and have to be fermented,
Aflatoxins are found as contaminants of food due to the devel- dried and roasted to obtain the characteristic cocoa flavor. During the
opment of fungi both pre-and post-harvest; the level of contamina- processing at farm, cocoa beans are exposed to bacteria, yeasts and
tion depends on the plant stress, temperature, humidity, genotype filamentous fungi present in the environment. Bacteria and yeasts
and culture and storage conditions (Wilson and Payne, 1994). An have an essential role in production of enzymes, acids and other
inhibition of their production has also been related to the presence of metabolites that take part in biochemical transformations of the
some food components such as caffeine and polyphenols (Lenovich beans, forming the precursors of chocolate flavor (Schwan and
and Hurst, 1979; Hasan, 1999; Molyneux et al., 2007). Wheals, 2004). On the other hand, despite the frequent reports of
Despite the ability of aflatoxin production detected in several filamentous fungi (Ribeiro et al., 1986; Ardhana and Fleet, 2003;
species of fungi: Aspergillus flavus, A. parasiticus, A. nomius, A. toxicarius, Mounjouenpou et al., 2008; Sanchez-Hervas et al., 2008; Copetti et al.,
A. parvisclerotigenus, A. bombycis, A. pseudotamarii, A. ochraceoroseus, submitted for publication), their function in the process is not clear.
A. rambelli, Emericella astellata and E. venezuelensis (Frisvad et al., Fungal presence in cocoa is generally regarded as undesirable and
2005); A. flavus and A. parasiticus remain as the most important and often related to the formation of off flavors, spoilage and mycotoxin
accumulation (Schwan and Wheals, 2004; Gilmour and Lindblom,
2008; Copetti et al., 2010).
⁎ Corresponding author. Tel.: + 55 55 3220 8822. In this study, we evaluated the occurrence and distribution of
E-mail address: mvc@smail.ufsm.br (M.V. Copetti). aflatoxigenic species through the stages of cocoa processing at the

0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2011.05.020
142 M.V. Copetti et al. / International Journal of Food Microbiology 148 (2011) 141–144

farm and their capacity to produce aflatoxins in culture medium, as 2.4.2. HPLC parameters
well as the occurrence of aflatoxins in these products, aiming to A Shimadzu LC-10VP HPLC system (Shimadzu, Japan) was used
correlate them in order to establish the critical steps in aflatoxin with a fluorescence detector set at 362 nm excitation and 455 nm
production on cocoa beans. emission for aflatoxins G1 and G2 and 425 nm emission for aflatoxins
B1 and B2. A Shimadzu CLC G-ODS (4 × 10 mm) guard column and
2. Materials and methods Shimadzu Shimpack (4.6 × 250 mm) column were employed. The
mobile phase used was water: acetonitrile: methanol (6:2:3, v/v/v)
2.1. Samples containing KBr (119 mg/L) and nitric acid (4 M, 350 μL/L). The flow
rate was 1 mL/min. A mix of aflatoxin B1, B2 G1 and G2 standard was
Two hundred and twenty six samples of cocoa beans correspond- used to construct a five point calibration curve of peak areas versus
ing to varieties of the groups Forastero and Trinitario were collected concentration (μg/mL). The injection volume was 100 μL for both
from farms in Bahia, the main cocoa producing region in Brazil. standard solution and sample extracts. The post-column derivatiza-
These samples were made up of 25 cocoa beans at the opening of the tion of aflatoxins B1 and G1 was performed with bromine using a
pods, 51 from different times of fermentation in wooden boxes (1 to KobraCell (R-Biopharm Rhône Ltd, Scotland).
6 days), 85 from different phases of sun drying on wooden floor
platforms with movable roofs (1 to 12 days) and 64 of dried beans in 2.4.1. Optimization of method performance
storage. Two different levels of contamination were tested to determine
the recovery of aflatoxins from cocoa using the above methodology,
2.2. Water activity determination in triplicate. Cocoa was spiked with a mix of aflatoxins containing 0.4,
0.2, 0.15 and 0.18 μg /kg; and 4.0, 2.4, 1.5 and 1.8 μg/kg respectively of
The water activity (aw) of cocoa bean samples was determined aflatoxin B1, B2, G1 and G2. For limit of detection (LOD) determination,
in triplicate in an Aqualab Series 3TE instrument (Decagon, USA) a concentration of aflatoxins close to an estimated detection limit was
at 25 ± 0.1 °C. spiked in 8 cocoa samples and the extractions were performed in
parallel. After quantification, the standard deviation was calculated
2.3. Identification of fungal species with the potential to produce and the limit of detection was determined according to recommen-
aflatoxins dations of Eurachem Guides (1998).

Samples (about 200 g) were subsampled (50 g) and surface 2.5. Statistical analyses
disinfected in a sodium hypochlorite solution (0.4%) for 2 min. A
total of 33 beans (eleven particles per plate) was placed aseptically on Statistical analyses (correlation analyses) were carried out with
Dichloran 18% Glycerol agar (DG18) (Pitt and Hocking, 2009). The the software The Unscrambler® 9.2 (Camo Process AS, Norway).
plates were incubated for 7 days at 25 °C. After incubation, beans were Correlation coefficients (r) were calculated to identify possible
inspected for fungal growth and all colonies isolated on Czapek Yeast associations between the incidence of fungi, the potential aflatoxin
Extract agar (CYA) (Pitt and Hocking, 2009) for subsequent producers and aflatoxin B1 levels (the most prevalent aflatoxin) in
identification of potentially aflatoxigenic fungi based both on the cocoa in all samples evaluated. Interpretation of values was
macroscopic (colony diameter, color, exudate and soluble pigment performed according to Pearson's coefficient (r) which are: very weak
production) and microscopic characters, following appropriate keys 0.000 ≤ r ≥ 0.200; weak 0.201 ≤ r ≥ 0.400; moderate 0.401 ≤ r ≥ 0.600;
(Klich and Pitt, 1988a; Samson et al., 2002). The isolation frequency of strong 0.601 ≤ r ≥ 0.800 and very strong 0.801 ≤ r ≥ 1.000 (Christmann
each species was expressed as the percentage of particles infected by and Badgett, 2009).
that species.
Fungi identified as potential producers of aflatoxins were inoc- 3. Results and discussion
ulated onto Yeast Extract Sucrose agar (Samson et al., 2002) for 7 days
at 25 °C and then the agar plug technique (Filtenborg et al., 1983) Cocoa processing on the farm resulted in a large reduction in aw
was used to evaluate the capability of isolates to produce aflatoxins. levels (Table 1). Most cocoa farms visited during this work used
Fungal extracts taken as plugs with a cork borer were placed on TLC empirical methods to decide when beans were dry enough to be
plates, developed in a toluene: ethyl acetate: formic acid 90%: stored. Our results indicate that the critical point for aflatoxigenic
chloroform (7:5:2:5, v/v/v/v) mobile phase, and visualized under fungi to infect cocoa beans is the sun drying stage, when the beans
UV light at 365 nm. A mixture of aflatoxins B1, B2, G1 and G2 standards start to lose water. The decrease of aw reduces the number of
(Sigma, St. Louis, USA) was used for comparison. competitors due to the high sensitivity of bacteria and yeasts to low
water availability (Beuchat, 1987). All the farms in this study used a
2.4. Analyses of aflatoxins on cocoa beans wooden drying floor, where it is difficult to maintain good hygienic
conditions and this can contribute to a high fungal load.
2.4.1. Clean-up Out of 604 isolates of A. flavus, 386 (63.9%) were producers of
Two g of NaCl were added to 20 g of finely ground cocoa and aflatoxins B1 and B2. The species A. parasiticus and A. nomius occurred
extracted with 120 mL of methanol: water solution (8:2, v/v). at a lower percentage, but all 212 isolates of A. parasiticus and the 3
Suspensions were blended (3 min) at high speed (10,000 rpm) A. nomius tested were able to synthesize aflatoxins B1, B2, G1 and G2.
using an Ultra-Turrax homogenizer (Polytron, Switzerland). The There is a big difference in the distribution of these species in foods
homogenized solution was filtered through Whatman No. 2 filter as well their capacity to produce aflatoxins. A. flavus has a wider
paper and Whatman A-H glass microfiber filter (Whatman, England). distribution and higher frequency of occurrence in foods when
The filtrate (4 mL) was diluted in phosphate buffered saline (24 mL) compared to A. parasiticus. However, according to literature, about
and applied to an Aflatest WB immunoafinity column (Vicam, USA) at half of the isolates of A. flavus are aflatoxigenic and this species
a flow rate of 2–3 mL/min. The column was then washed with distilled produces only aflatoxins from the class B, while about 100% of
water (30 mL), and aflatoxins eluted with methanol (4 mL) into an A. parasiticus have such ability, and synthesize aflatoxins from groups
amber vial. After evaporation to dryness at 40 °C under a stream of N2, B and G (Klich and Pitt, 1988b; Vaamonde et al., 2003).
the dry residue was redissolved in methanol:water (2:3, v/v; 1 mL) A. flavus was present in samples from all stages after the beginning
and filtered through Millex PTFE 0.45 μm (Millipore, USA). of cocoa fermentation. About 40% of the samples analyzed from sun
 M.V. Copetti et al. / International Journal of Food Microbiology 148 (2011) 141–144 143

Table 1
Water activity and aflatoxin contamination in cocoa beans at different processing stages at farm.a

Stage (n) aw Aflatoxins (μg/kg)

B1 B2 G1 G2 Total

Before fermentation (25)

Mean 0.99 b LOD bLOD bLOD b LOD b LOD
Range 0.98–0.99 b LOD bLOD bLOD b LOD b LOD
Positive (NLOD) – 0 0 0 0 0

Fermentation (51)
Mean 0.99 b LOD bLOD bLOD b LOD b LOD
Range 0.98–0.99 b LOD–0.1 bLOD–0.04 bLOD–0.06 b LOD b LOD–0.2
Positive (NLOD) – 2 (4%) 1 (2%) 1 (2%) 0 2 (4%)

Sun drying (85)

Mean 0.81 0.11 0.02 bLOD b LOD 0.13
Range 0.49–0.99 b LOD–6.66 bLOD–0.37 bLOD b LOD b LOD–7.03
Positive (NLOD) – 11 (13%) 4 (4%) 0 0 11 (13%)
Positive (N1 μg/kg) – 2 (2.4%) 0 0 0 2 (2.4%)

Storage (65)
Mean 0.67 b LOD bLOD bLOD b LOD b LOD
Range 0.40–0.85 b LOD–0.14 bLOD bLOD b LOD b LOD–0.14
Positive (NLOD) – 3 (5%) 0 0 0 3 (5%)
LOD – 0.001 0.002 0.003 0.02
a
Method mean recovery: 94.79%.

drying were contaminated, reaching 100% of infected beans in some At the stage of sun drying on platforms, only 11 (13%) samples
samples. During storage the contamination by A. flavus remained were contaminated with aflatoxins (Table 1). From these, two
high (32% of infected samples) and so did the number of infected samples presented total aflatoxin levels of 0.1 and 0.5 μg/kg, 3
beans in each sample, which also reached 100% in some cases samples exceeded 0.5 μg/kg, one of these reaching 6.66 μg/kg
(Table 2). aflatoxin B1. A. flavus was isolated in the 3 samples with higher levels
Compared to A. flavus, the isolates of A. parasiticus appeared to be of aflatoxins and A. parasiticus was co-occurring in one sample. This
more sensitive to aw reduction. This species was isolated from about low level of aflatoxins in the samples was unexpected given the high
25% of the samples at sun drying and only 14% during storage. A frequency of occurrence of aflatoxigenic fungi in the samples, and it
reduction in the number of infected beans was also observed, which can be visualized through Pearson's correlation coefficient values
reached 78% infection during drying and no more than 48% in storage analyses (Table 3).
(Table 2). In this regard, these important aflatoxigenic species may There was a weak correlation between contamination by aflatoxin
show different physiological patterns. B1 and presence of aflatoxigenic fungi (both A. flavus or A. parasiticus),
There are differences between the minimum aw of a substrate to suggesting the existence of anti-toxigenic properties in cocoa, limiting
allow fungal growth and that which is necessary for mycotoxin the accumulation of aflatoxins in this product (Table 3).
production; the latter is generally more restrictive. The growth of The action of polyphenols on the synthesis and accumulation of
A. flavus and A. parasiticus has been verified with a minimum aw of aflatoxins in food has been observed by Molyneux et al. (2007). They
0.78 but a minimum aw of 0.82 and 0.86 is required for A. flavus and evaluated the effect of different phenolic constituents commonly
A. parasiticus to produce aflatoxins, respectively (Pitt and Hocking, present in oilseeds (walnuts, almonds and pistachios) on mycotoxin
2009). These aw values were still found at the sun drying stage production and found inhibition between 59.5 and 99.8% of the
(Table 1). synthesis of aflatoxin. It is known that cocoa is a product rich in
Among the samples collected before the beginning of fermentation polyphenols (Wollgast and Anklam, 2000; Lee et al., 2003), which
and during the fermentation, none showed any contamination by could act as inhibitors of aflatoxins in this product.
aflatoxins. Table 1 shows the results of aflatoxin analyses in samples A study conducted by Hasan (1999) evaluated the role of caffeine
collected during the primary processing of cocoa beans on the farm. and tannin (a polyphenol) in anti-toxigenic properties of coffee and
tea, since A. flavus is a common contaminant of these products,
and the amount of aflatoxin produced in these products was very low.
The researcher found a 5-fold increase in aflatoxin production in
detannin-caffeinated coffee and tea compared with normal, when
Table 2 inoculated with A. parasiticus. The addition of tea extract to a culture
Isolation frequency of aflatoxigenic species and incidence of infected cocoa beans at
different processing stages.a

Stage Fermentation Drying Storage

(n) (51 samples) (85 samples) (65 samples) Table 3

Correlation coefficients (r) of aflatoxigenic fungi, water activity and aflatoxin B1.
IF (%) RI (%) IF (%) RI (%) IF (%) RI (%)
Parameter Aflatoxin B1 aw
Aspergillus flavus 3.92 ND-6 39.51 ND-100 32.31 ND-100
Aspergillus nomius 0 ND 3.70 ND-3 0 ND Aflatoxin B1 1.000 0.004
Aspergillus parasiticus 1.96 ND-6 25.92 ND-78 13.85 ND-48 Water activity 0.004 1.000
Total fungi contamination (all fungi isolated) 0.085 −0.292
RI = Range of Infection % (Range of infected beans in a sample, %).
a Aspergillus flavus 0.383 −0.270
IF = Isolation frequency % (number of samples containing a fungal species/ total of
Aspergillus parasiticus −0.270 −0.003
samples evaluated, %).
144 M.V. Copetti et al. / International Journal of Food Microbiology 148 (2011) 141–144

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