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Review
Fungi and mycotoxins in cocoa: From farm to chocolate
Marina V. Copetti
a,
⁎, Beatriz T. Iamanaka
b
,JohnI.Pitt
c
, Marta H. Taniwaki
b
a
Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brazil
b
Instituto de Tecnologia de Alimentos, Campinas, SP 13070-178, Brazil
c
CSIRO Animal, Food and Health Sciences, North Ryde, NSW 2113, Australia
abstractarticle info
Article history:
Received 24 October 2013
Received in revised form 13 February 2014
Accepted 27 February 2014
Available online 11 March 2014
Keywords:
Ochratoxin A
Aflatoxin
Chocolate
Cocoa by-products
Food safety
Cocoa is an important crop, as it is the raw material from which chocolate is manufactured. It is grown mainly in
West Africaalthough significantquantities also come fromAsia and Central and South America. Primary process-
ing is carried out on the farm, and theflavour of chocolate starts to develop at that time. Freshly harvested pods
are opened,the beans, piled in heapsor wooden boxes, are fermented naturallyby yeasts and bacteria,then dried
in the sun on wooden platforms or sometimes on cement or on the ground, where a gradual reduction in mois-
ture content inhibits microbial growth. Beans are then bagged and marketed. In processing plants, the dried
fermented beans are roasted, shelled and ground, then two distinct processes are used, to produce powdered
cocoa or chocolate. Filamentous fungi may contaminate many stages in cocoa processing, and poor practices
may have a strong influence on the quality of the beans. Apart from causing spoilage, filamentous fungi may
also produce aflatoxins and ochratoxin A. This review deals with the growth of fungal species and formation of
mycotoxins during the various steps in cocoaprocessing, as wellas reduction of these contaminants bygood pro-
cessing practices. Methodologies for fungal and mycotoxin detection and quantification are discussed while cur-
rent data about dietary exposure and regulation are also presented.
© 2014 Elsevier B.V. All rights reserved.
Contents
1. Introduction............................................................... 14
2. Fungiandmycotoxinsinfarmprocessingofcocoa.............................................. 14
2.1. Fermentation........................................................... 14
2.2. Drying.............................................................. 15
2.3. Storage.............................................................. 16
3. Fungiandmycotoxinsincocoamanufacturing................................................ 16
3.1. Roasting ............................................................. 16
3.2. Shellingandwinnowing...................................................... 16
3.3. Grindingorconching........................................................ 17
3.4. Powderedcocoamanufacture.................................................... 17
3.5. ChocolateManufacturing...................................................... 17
4. Methodologyfordetectionoffungiandmycotoxinsincocoaandcocoaproducts................................ 17
4.1. Fungi............................................................... 17
4.2. Mycotoxins............................................................ 18
5. Mycotoxinexposureandregulationincocoaandcocoaproducts....................................... 18
6. Preventionofmycotoxinformationincocoa................................................. 18
7. Finalconsiderations............................................................ 19
References .................................................................. 19
International Journal of Food Microbiology 178 (2014) 13–20
⁎Corresponding author at: Departamento de Tecnologia e Ciência de Alimentos, Centro de Ciências Rurais, Universidade Federal de Santa Maria, Santa Maria, RS 97105-900, Brazil.
Tel.: +55 55 3220 8254x209.
E-mail address: mvc@smail.ufsm.br (M.V. Copetti).
http://dx.doi.org/10.1016/j.ijfoodmicro.2014.02.023
0168-1605/© 2014 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
International Journal of Food Microbiology
journal homepage: www.elsevier.com/locate/ijfoodmicro
Author's personal copy
1. Introduction
Cocoa is an important crop, as it is the raw material from which
chocolate is manufactured. Cocoa trees thrive best in very humid tropi-
cal climates, and are grown mainly in West Africa although significant
quantities also come from Asia and Central and South America. Primary
processing is carried out on the farm. Pods are harvested and opened by
hand. The beans are piled in heaps or in wooden boxes, and allowed to
undergo a natural fermentation. Microorganisms contaminate the
beans from the outer surfaces of pods, workers' hands and tools, plant
leaves, collection baskets, insects or residual mucilage in equipment.
The desirable types are yeasts, and lactic or acetic acid bacteria, which
secrete enzymes, alcohol and lactic and acetic acids (Ostovar and
Keeney, 1973; Schwan and Wheals, 2004). These metabolic products
lead to embryo death and production of important precursors of choco-
late aroma (Voigt, 2013). After fermentation, which lasts several days,
the beans are transferred to wooden sun drying platforms, or some-
times are dried on cement or on the ground, where a gradual reduction
in moisture content and volatile acid production occurs, eventually
stopping microbial growth and enzyme production. When the beans
are fully dry, they are transferred to storage rooms, then later bagged
and marketed. In processing plants, the dried fermented cocoa beans
are roasted, shelled and ground, then two distinct processing lines pro-
duce powdered cocoa or chocolate.
Cocoa beans are susceptible to fungal contamination during many of
these processing steps. Microbial growth is affected by intrinsic param-
eters of cocoa beans such as pH,by water activity and by the various or-
ganic acidsproduced during fermentation. Besides causing deteriorative
alteration of sensorial properties, the presence of filamentous fungi in
cocoa and chocolate is also a cause for concern due to the possibility
of mycotoxin formation. Both aflatoxin and ochratoxin A have been re-
ported from cocoa and chocolate.
The discovery of ochratoxin A in cocoa and by-products prompted
international discussions. In 2012 the Codex Committee on Contami-
nants in Food elaborated a discussion paper on ochratoxin A in cocoa
(Codex Alimentarius Commission, 2012). Information was gathered
on the occurrence of ochratoxin A in cocoa and by-products to deter-
mine levels of contamination, the contribution of these products tooch-
ratoxin A in the human diet,to elucidate the main factors responsible for
ochratoxin A synthesis in cocoa and to reduce it during processing. A
code of practice was formulated and is under discussion (Codex
Alimentarius Commission, 2013).
This review focuses on the factors affecting the development of fila-
mentous fungi and the potential for formation of aflatoxins and ochra-
toxin A at all processing stages. An update on international reports of
mycotoxins in cocoa and chocolate is also provided.
2. Fungi and mycotoxins in farm processing of cocoa
2.1. Fermentation
Beans and pulp inside an intact, healthy cocoa pod are microbiolog-
ically sterile (Fig. 1,1A–C) but when opened soon become contaminated
with microorganisms, especially those that will contribute to the subse-
quent natural fermentation process.
During fermentation, the microbial population is dominated by
yeasts in the first hours after which their level is surpassed by those of
lactic acid bacteria, that in turn decline after 48 h of fermentation after
which acetic acid bacteria develop, in a well established pattern of suc-
cession (Schwan and Wheals, 2004; Nielsen et al., 2013). Lima et al.
(2011) present in detail the importance of fermentation and environ-
mental factors in the development of a good quality product. The prod-
ucts of microbial metabolism strongly influence the microbial
population that sequentially dominate this micro-environment. High
amounts of alcohol are produced by yeasts and lactic and acetic acids
by bacteria. Together with environmental factors including low pH,
the action ofthe organic acids, elevated temperatures due to exothermic
reactions and microaerophilic conditions, these fermentation products
restrict the growth of filamentous fungi. Studies reported the presence
of filamentous fungi especially in the last days of fermentation, in the
surface (Fig. 1, 2A) (Schwan and Wheals, 2004; Copetti et al., 2013a)
or when the cocoa mass is not turned regularly(Nielsen et al., 2013). Fil-
amentous fungi can sometimes be seen in the cocoa inside the fermen-
tation boxes when the mass is turned (Fig. 1, 2B).
The role of filamentous fungi during cocoa fermentation is not well
understood. It is known that some species can cause hydrolysis of the
pulp, produce acids or off flavours and so alter the taste of the cocoa
beans (Ardhana and Fleet, 2003; Schwan and Wheals, 2004). Extensive
fungal development at the end of fermentation may cause increased de-
terioration in the drying phase (Gilmour and Lindblom, 2008).
After studying the microbial ecology of cocoa fermentation in wooden
boxes in Indonesia, Ardhana and Fleet (2003) observed the presence of
Penicillium citrinum and an unidentified basidiomycete in the first 36 h
of fermentation. Both fungi showed strong polygalacturonase activity,
suggesting their role in the degradation of pulp in the early stages of
fermentation. The presence of Aspergillus versicolor,Aspergillus wentii
and Penicillium purpurogenum was also reported (Ardhana and Fleet,
2003).
Fig. 1. 1A, cocoa pod; 1B, cocoa beanssurrounded by pulp;2A, 2B, fungal presence during
fermentation; 3A, sun drying of cocoa beans; 3B, mouldy cocoa beans at drying; 4A, stor-
age of cocoa beans; 4B, mouldy cocoa beans in storage; 1C, 2C, 3C, 4C, mycological evalu-
ation of cocoa beans by direct plating in DG18, 1C, before fermentation; 2C, during
fermentation; 3C, during drying; 4C, during storage.
14 M.V. Copetti et al. / International Journal of Food Microbiology 178 (2014) 13–20
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The occurrence of filamentous fungi, especially species potentially
producing ochratoxin A, was compared between heap and box fermenta-
tion in Cameroon by Mounjouenpou et al. (2008). No significant differ-
ences were seen between the two methods in relation to the fungal
species found (Aspergillus fumigatus,Aspergillus tamarii,A. versicolor,As-
pergillus carbonarius,Aspergillus niger,Penicillium sclerotiorum,Penicillium
paneum,Penicillium crustosum,Mucor spp., Rhizopus spp., Fusarium spp.
and Trichoderma spp.). However, pod integrity and, to a lesser degree, a
delay in pod opening, affected fungal diversity. Damaged pods often
showed proliferation of toxigenic fungi including A. carbonarius,A. niger,
with the potential to produce ochratoxin A, and Fusarium species.
In a study of cocoa fermentation in Brazil (Copetti et al., 2011a), 18
species of filamentous fungi were isolated. However, only Monascus
ruber,P. paneum and Geotrichum candidum were present in more than
20% of the 51 samples examined, 19.6, 23.5 and 25.5%, respectively,
with an average of beans infected of 14.5, 13.7 and 46.6%, respectively
(Fig. 1, 2C). Existing physiological information (Pitt and Hocking, 2009)
provided suggestions that might explain the higher prevalence of these
species. G. candidum is able to grow only at high water activities, but
under microaerophilic conditions, while M. ruber and P. paneum are capa-
ble of growth under low oxygen tension. P. paneum is closely related to
Penicillium roqueforti, known to tolerate high levels of CO
2
and weak
acid preservatives similar to those found in cocoa fermentation. Other
acetic acid tolerant species, Paecilomyces variotii and Thielaviopsis
ethaceticus, have been reported from cocoa fermentations on Brazilian
farms (Ribeiro et al., 1986).
Species producing aflatoxins, Aspergillus flavus and Aspergillus
parasiticus, have been isolated in samples from fermentations (Copetti
et al., 2011a), and also A. niger and A. carbonarius, species producing
ochratoxin A (Mounjouenpou et al., 2008; Copetti et al., 2010). In
general mycotoxin producing species were present in less than 5% of
samples during fermentation (Copetti et al., 2010, 2011a), but these ini-
tial inocula could contribute to elevated populations when competition
diminishes in subsequent processing steps.
Only a few samples of cocoa collected during fermentation have been
analysed for mycotoxins: three studies for ochratoxin A (Gilmour and
Lindblom, 2008; Mounjouenpou et al., 2008; Copetti et al., 2010), and
one for aflatoxins (Copetti et al., 2011a). While mycotoxin formation
was found to be possible during fermentation, reported concentrations
were low, on average lower than 0.02 μg/kg for aflatoxins and about
0.05 μg/kg for ochratoxin A when healthy pods were sampled (Gilmour
and Lindblom, 2008; Mounjouenpou et al., 2008; Copetti et al., 2010,
2011a).
2.2. Drying
At the end of fermentation, cocoa beans contain 40–60% moisture
and should be dried to 6–7% moisture for microbial stability. Drying
may be carried out in the sun on wooden platforms (Fig. 1,3A),inme-
chanical driers: sometimes both methods are used under changeable
weather conditions. Sun drying, the common method, usually takes
about 7 days under sunny conditions but can take 2 to 4 weeks if the
weather is adverse. As would be expected, prolonged drying increases
the chance of fungi growth and spoilage (Nielsen et al., 2013).
As the water activity is reduced during drying, from a water activity
of 0.99 down to 0.85, first the growth of bacteria ceases, then that of the
yeasts, which have a higher tole rance of low wat er availability (Beuchat,
1987). Xerophilic fungi become dominant in the later stages of drying,
as water activity continues to decrease (Fig. 1,3B–C). The final water ac-
tivity of the beans is about 0.50 (Copetti et al., 2010). Wooden drying
Fig. 2. The industrial processingof cocoa beans and main steps where the reduction of mycotoxin contamination occurs.
15M.V. Copetti et al. / International Journal of Food Microbiology 178 (2014) 13–20
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platforms are a source of fungal spore inoculum. Sun drying in thin
layers also increases the oxygen tension, enhancing filamentous fungal
growth and at the same time reducing the concentration of inhibitory
acids produced during the fermentation, especially acetic acid, due to
volatilisation (Copetti et al., 2012a).
Yeasts and fungal species established during fermentation dominate
during the first days of drying, but are overtaken by genera adapted to
lower moisture, especially Aspergillus and Penicillium (Copetti, 2009).
Numbers of potentially toxigenic species, including A. flavus,
A. parasiticus,A. niger and A. carbonarius, increase during the later stages
of drying (Fig. 1,3C)(Copetti et al., 2011b).
Frequent isolation of species potentially producing aflatoxin from
samples during drying is a cause for concern (Copetti et al., 2011a).
Water activities often remain high for long enough to permit mycotoxin
production (Copetti et al., 2011b). However, concentrations of afla-
toxins found in samples evaluated were very low (mean 0.13 μg/kg),
suggesting the existence of inhibitors in cocoa. Inhibition could be relat-
ed to the presence of high levels of polyphenols reported in oilseeds by
Molyneux et al. (2007), who observed up to 99.8% inhibition of aflatoxin
synthesis in the presence of these antioxidants.
About 50% of samples taken during sun drying were positive for och-
ratoxin A (Copetti et al., 2010), but levels were generally low, with a
mean of only 0.13 μg/kg. A correlationwas observed betweenthe occur-
rence of ochratoxin A in the samples and the presence of A. carbonarius,
indicating that it is the major species producing ochratoxin A contami-
nation in cocoa.
2.3. Storage
Dried beans are typically stored in bags at farms until marketed
(Fig. 1,4A–B) (Minifie, 1999). As spores of the fungi present at the end
of drying (Fig. 1, 4B) remain viable for long periods, good storage condi-
tions are crucial to maintaining the quality of the beans. As cocoa beans
are low in soluble solids, storage at high humidity may cause rapid in-
creases in water activity, providing suitable conditions for spore germi-
nation, fungal growth and spoilage (Raters and Matissek, 2003). Wood
(1985) recommended that only under carefully controlled conditions
should cocoa storage in tropical countries exceed 2–3months.
Studies have reported toxigenic fungal species, usually A. flavus and
A. niger, in dried cocoa beans from all growing areas (Niles, 1981;
Aroyeun et al., 2007; Mounjouenpou et al., 2008; Rahmadi and Fleet,
2008; Sanchez-Hervas et al., 2008; Copetti et al., 2011b). Xerophilic spe-
cies, especially Eurotium amstelodami,Eurotium chevalieri,Eurotium
rubrum and Aspergillus penicillioides will also grow when cocoa beans
are stored under poor conditions (Rahmadi and Fleet, 2008; Copetti
et al., 2011a)(Fig. 1, 4C). These species grow under conditions of re-
duced water activity and are responsible for large economic losses in
stored grains, nuts, spices and cereal products (Pitt and Hocking,
2009). However, these species produce no significant mycotoxins.
Aflatoxins were first reported in cocoa by Campbell (1969). Most
stored samples show low contamination (Raters and Matissek, 2003;
Aroyeun et al., 2007; Copetti et al., 2011a). Concentrations of ochratoxin
A in cocoa beans appear to vary according to origin, with higher levels in
samples coming from Ivory Coast (Raters and Matissek, 2003; Gilmour
and Lindblom, 2008), and sometimes Nigeria (Dongo et al., 2008). A
high proportion of cocoa bean samples have been reported to be con-
taminated with ochratoxin A but in most studies fewer than 20% of sam-
ples had concentrations above 2 μg/kg (Amezqueta et al., 2004;
Bonvehi, 2004; Gilmour and Lindblom, 2008; Dembele et al., 2009;
Copetti et al., 2010; de Magalhaes et al., 2011).
3. Fungi and mycotoxins in cocoa manufacturing
Cocoa beans are manufactured into powdered cocoa or chocolate by
a series of processing steps, involving heat treatment or segregation of
fractions (Fig. 2), which impact on fungal and mycotoxin contamination
in finished products. The influence of the various factors leading to my-
cotoxin formation and reduction in cocoa fermentation, drying and pro-
cessing are shown in Fig. 3. Mycotoxin concentrations reported from
cocoa and cocoa products are shown in Table 1.
3.1. Roasting
Roastingcompletes the chemical reactions responsible for the devel-
opment of chocolate aroma and is a critical step for ensuring cocoa qual-
ity (Voigt, 2013). At the same time this process reduces microbial
contamination. Roasting nibs, with treatments of 15 min to 2 h at
105–150 °C, is considered to be the only step in chocolate production
that destroys all microorganisms (ICMSF, 2005).
Roasting decreased ochratoxin A concentrations by 24–40% in ex-
periments conducted by Manda et al. (2009), while the reduction was
about 17% under conditions evaluated by Copetti et al. (2013b).
3.2. Shelling and winnowing
The cocoa bean testa (shell) makes up about 12% of the weight in
fermented and dried cocoa beans. A maximum of only 1.0–1.5% of
testa residues is generally allowed in the nibs (beans), so shelling is
followed by winnowing (Minifie, 1999). Most contaminant fungi are
present in the testa, especially Aspergillus,Eurotium and Absidia species
(Copettiet al., 2010) and are almost completely removed by winnowing
(Minifie, 1999; Copetti et al., 2010).
It has been reported that ochratoxin A is conce ntrated in the testa frac-
tion, so that only a small part of the toxin contaminates the nibs (Bonvehi,
2004; Amezqueta et al., 2005; Gilmour and Lindblom, 2008; Manda et al.,
2009; Copetti et al., 2013b). Mechanical shelling removed an average of
48% (range 27–72%) of ochratoxin A (Gilmour and Lindblom, 2008),
while shelling by hand reduces between 50 and 100% (Amezqueta et al.,
2005; Manda et al., 2009). The efficiency of winnowing is therefore a crit-
ical point for reducing the level of contaminants present in the nibs before
the subsequent processing steps (Copetti et al., 2013b).
Fig. 3. Schematic of formation and reduction in aflatoxin and ochratoxin A duringstages of production of cocoa powder and chocolate from cocoa beans. The diagram is qualitative.
16 M.V. Copetti et al. / International Journal of Food Microbiology 178 (2014) 13–20
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3.3. Grinding or conching
Nibs are then ground using large oscillating stones known as
conches at temperatures of 50–70 °C to form cocoa liquor, also called
cocoa mass when cool. Grinding requires 2 to 72 h, depending on the
equipment type, cocoa quality and desired chocolate type (Beckett,
2008). At this point the processing can follow two different ways, to
produce either powdered cocoa or chocolate.
3.4. Powdered cocoa manufacture
Powdered cocoa is manufactured by pressingcocoa mass under high
pressure and temperatures between 95 and 105 °C. Separation out of
cocoa butter leaves a residual cake, which has relatively little fat (12–
22%) and high solids (Minifie, 1999). The cake is then milled to produce
“natural”cocoa powder (Fig. 2).
In the pressing step, ochratoxin A remains bound to the cocoa cake
(Table 1), so levels found in the butter are very low. Higher levels of
cocoa solids non fat in a final product tend to be correlated with higher
concentrations of ochratoxin A in it (Miraglia and Brera, 2002; Bonvehi,
2004; Gilmour and Lindblom, 2008; Copetti et al., 2013b; Turcotte et al.,
2013). The same correlation has been observed for aflatoxins (Copetti
et al., 2012a; Turcotte et al., 2013).
The final step in producing powdered cocoa is an alkali process, usual-
ly using potassium carbonate in combination with heat. This process in-
fluences the dispersibility of the cocoa particles in liquids and is an
important influence in the final colour of the cocoa powder (Minifie,
1999).
The alkali process produces a sterile product, nevertheless fungi can
be isolated from cocoa products sometimes. These fungi are likely to be
postprocessing contaminants (Copetti et al., 2011a).
Mycotoxin levels in alkalized cocoa powder tend to be lower than in
untreated fractions (Copetti et al., 2011b, 2013b; Turcotte et al., 2013),
although the alkali process appears to be more effective in reducing af-
latoxin concentrations than those of ochratoxin A.
3.5. Chocolate Manufacturing
Chocolate is the homogeneous combination of cocoa materials (li-
quor and butter) with milk products, sugars and/or sweeteners, and
other additives (Codex Alimentarius Commission, 2003). The addition
of these other ingredients dilutes the mycotoxin content of the final
product but may also introduce new contaminants. The homogenising
process is carried out at temperatures between 45 and 100 °C, causing
a decrease in acidity and moisture content, enhancement of the Maillard
reaction and a reduction in microbial contamination.
Chocolate is regarded as a microbiologically stable product because of
its low water activity. However, extremely xerophilic fungi such as Bettsia
alvei,Chrysosporium xerophilum and Xeromyces bisporus can cause deteri-
oration in chocolate and chocolate confectionery (ICMSF, 2005;
Kinderlerer, 1997). Storage under high humidity may be partly responsi-
ble. The xerophilic yeast Zygosaccharomyces rouxii sometimes causes leak-
er spoilage in filled chocolates. The water activity in chocolates is not
sufficiently low to prevent growth of this yeast, so chocolate fillings
must be free of it during chocolate manufacture (Pitt and Hocking,
2009). Mycotoxigenic fungal species have been not reported in chocolate.
Ochratoxin A in chocolate was first reported by Engel (2000).Since
that time, a considerable number of chocolate samples have been eval-
uated and the majority have been reported as positive (Table 2). Con-
centrations are generally low. As noted above, the concentration of
ochratoxin A tends to be directly related to the amount of cocoa solids
used in the chocolate formulation.
The literature has few reports on aflatoxins in chocolate; these are
summarised in Table 2.Kumagai et al. (2008) reported that 50% of sam-
ples of bitter chocolate analysed were contaminated with aflatoxin B
1
.
The occurrence of both aflatoxins and ochratoxin A in at least 80% of choc-
olate samples was reported by Copetti et al. (2012b) in Brazil and by
Turcotte et al. (2013) in Canada (Table 2). It is possible that cocoa butter
substitutes may be an additional source of aflatoxins in chocolate
(Kershaw, 1982).
Overall, it has been reported that processing, from the unroasted
bean to manufactured chocolate, results in more than 90% reduction
in ochratoxin A concentrations, mostly as the result of shelling.
4. Methodology for detection of fungi and mycotoxins in cocoa and
cocoa products
4.1. Fungi
The choice of plating technique and medium will influence results of
mycological analyses in all types of foods, including cocoa and cocoa
products. The International Commission on Food Mycology (ICFM) rec-
ommends direct plating as the preferred technique for solid products
such as chocolate. Surfacedisinfection of pieces in 0.4% freshly prepared
chlorine(household bleach) before directplating is considered essential
(Hocking et al., 2006). However, analyses are more commonly carried
out on powders, and here ICFM recommends dilution plating. Initial di-
lution 1:9 in 0.1% peptone with homogenisation in a Stomacher is
Table 1
Occurrence of ochratoxin A in samples of cocoa products.
Cocoa product Ochratoxin A Origin Reference
Number of samples evaluated Positive N2μg/kg Mean (μg/kg) Maximum (μg/kg)
Beans 33 24 5 1.85 14.8 Ivory Coast Amezqueta et al. (2004)
Beans 7 3 –1.55 3.88 Cameroon Amezqueta et al. (2004)
Beans 6 2 0 0.26 0.42 Equatorial Guinea Amezqueta et al. (2004)
Beans 29 24 0 0.1 0.38 Brazil Copetti et al. (2013a,b)
Beans 21 16 1 0.45 3.5 West Africa Bonvehi (2004)
Butter 4 0 0 b0.1 b0.1 West Africa Bonvehi (2004)
Butter 25 20 0 0.03 0.05 Brazil Copetti et al. (2013a,b)
Butter 5 –0 0.03 0.08 –Turcotte et al. (2013)
Cake 80 74 41 2.79 9 Various Bonvehi (2004)
Cake 26 26 –0.97 3.18 Brazil Copetti et al. (2013a,b)
Liquor/mass 8 4 2 1.07 3.5 West Africa Bonvehi (2004)
Liquor/mass 25 25 0 0.34 1.09 Brazil Copetti et al. (2013a,b)
Liquor/mass 5 5 0 0.43 0.56 –Turcotte et al. (2013)
Nibs 2 0 0 b0.1 b0.1 West Africa Bonvehi (2004)
Powder (alkalized) 28 28 –0.9 3.59 Brazil Copetti et al. (2013a,b)
Powder (alkalized) 21 21 0 1.06 1.88 –Turcotte et al. (2013)
Powder (natural) 16 16 –1.42 5.13 Brazil Copetti et al. (2013a,b)
Powder (natural) 15 15 2 1.17 4.72 –Turcotte et al. (2013)
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recommended (Hocking et al., 2006). ICFM recommends Dichloran
Rose Bengal Chloramphenicol agar (DRBC; King et al., 1979) as a general
purpose isolation and enumeration medium for foods of high water ac-
tivity, i.e. N0.95 and Dichloran 18% Glycerol agar (DG18; Hocking and
Pitt, 1980) for analyses of food with water activities b0.95 (Hocking
et al., 2006). However, for analyses of chocolate, the medium of choice
is malt yeast 50% glycerol agar (Pitt and Hocking, 2009), as only extreme
xerophiles are able to cause spoilage of chocolate.
Sanchez-Hervas et al. (2008) analysed cocoa beans by direct plating,
using DRBC as medium, while Amezqueta et al. (2008) and
Mounjouenpou et al. (2008) used potato dextrose agar and dilution plat-
ing. The most recent study by Copetti et al. (2011a,b) used direct plating
and DG18.
4.2. Mycotoxins
Levels of mycotoxins in cocoa and chocolate are usually very low, so
sensitive methods should be used for their detection. Sampling and
analysis are both of critical importance to avoid unacceptable consign-
ments being accepted or satisfactory loads being unnecessarily rejected
(Turner et al., 2009). For ochratoxin A and aflatoxins in cocoa and by-
products, the common technique is high performance liquid chroma-
tography (HPLC) with fluorescence detection (FLD), because these my-
cotoxins fluorescence naturally, enabling very low detection limits.
The combination of the HPLC-FLD method with cleanup by immuno-
affinity columns has been used for the detection of ochratoxin A in var-
ious studies on cocoa beans (Amezqueta et al., 2004; Bonvehi, 2004;
Copetti et al., 2010; de Magalhaes et al., 2011), cocoa products (Brera
et al., 2003, 2011; Manda et al., 2009; Bonvehi, 2004; Turcotte et al.,
2013) and chocolate (Bonvehi, 2004; Manda et al., 2009; Brera et al.,
2011; Copetti et al., 2012b; Turcotte et al., 2013). This methodology
has also been the choice for aflatoxin analyses (Kumagai et al., 2008;
Copetti et al., 2011a, 2012b; Turcotte et al., 2013). The values found dur-
ing protocol validation were similar for ochratoxin A and aflatoxins,
showing recoveries of about 90% and limit of detection close to
0.05 μg/kg in most reports.
Recently the use of tandem mass spectrometry (LC/MSMS) to ana-
lyse mycotoxins in foods has increased, the main advantage being the
detection and quantification of mycotoxins simultaneously. However
methodologies developed to evaluate mycotoxins in cocoa or cocoa
products are not available. Besides that the cost of the equipment and
maintenance are still high, so HPLC-FLD is still the most commonly
used instrument for analysing mycotoxins in cocoa and cocoa products.
5. Mycotoxin exposure and regulation in cocoa and cocoa products
A survey of dietary intake of ochratoxin A concluded that cocoa rep-
resents about 5% of ochratoxin A intake in thediet of the European pop-
ulation (Miraglia and Brera, 2002). Chocolate was reported to
contribute about 6% of the total dietary exposure to ochratoxin A
(Codex Alimentarius Commission, 2012).
Few countries have set regulatory limits for ochratoxin A and/or af-
latoxins in cocoa beans and cocoa products. An expert panel of the
European Union recommended setting a maximum limit for ochratoxin
Aof1μg/kg in chocolate, chocolate powder and drinking chocolate and
2μg/kg in cocoa beans, cocoa nibs, cocoa mass, cocoa cake and cocoa
powder (Tafuri et al., 2004). However, the European Commission has
stated that a maximum limit for ochratoxin A in cocoa and cocoa prod-
ucts does not appear necessary (European Commission, 2010).
On the basis of significant consumption of cocoa products and choc-
olate by children and results published by Copetti et al. (2010, 2011a),
the Brazilian Sanitary Surveillance Agency (ANVISA) set limits of
10 μg/kg for cocoa beans and 5 μg/kg for cocoa products and chocolate
sold in Brazil, for both ochratoxin A and total aflatoxins (ANVISA, 2011).
6. Prevention of mycotoxin formation in cocoa
Mycotoxins are stable compounds in storage, and are more or less
resistant to chemical and physical treatments, so the best approach to
limiting mycotoxin contamination in foods is reduction of formation.
A code of practice to assist in preventing the formation and improve
the reduction of ochratoxin A in cocoa is under discussion (Codex
Alimentarius Commission, 2013).
In a study focused on the Ivory Coast, the main factors involved in the
occurrence of ochratoxin A in cocoa were elucidated by Gilmour and
Lindblom (2008). They reported that ochratoxin A formation may com-
mence between harvest and fermentation, especially with small holders,
and that damaged pods appear to be critical for ochratoxin A accumula-
tion, an observation confirmed by Mounjouenpou et al. (2008). To mini-
mize the problem, the Codex Alimentarius Commission (2013) has
Table 2
Occurrence of total aflatoxins and/or ochratoxin A in chocolate bars.
Product Aflatoxin, total Ochratoxin A Origin Reference
Number of
samples
Positive N1μg/kg Max. (μg/kg) Mean (μg/kg) Positive N1μg/kg Max. (μg/kg) Mean (μg/kg)
Bitter/dark 78 78 –
a
1.8 0.59
b
Germany Engel (2000)
Bitter 42 22 0 0.60 0.18 –– – – Japan Kumagai et al. (2008)
Bitter 41 –– – – 27 0 0.94 0.35 Japan Kumagai et al. (2008)
Bitter 25 25 4 1.65 0.66 25 0 0.60 0.31 Various Copetti et al. (2012b)
Dark 35 –– – – 35 –– 0.25
b
Spain Burdaspal and Legarda (2003)
Dark 52 –– – – 52 –– 0.27
b
Various Burdaspal and Legarda (2003)
Dark 536 –– – – 536 4 –0.26 Europe Gilmour and Lindblom (2008)
Dark 25 25 0 0.91 0.43 25 0 0.87 0.34 Brazil Copetti et al. (2012b)
Dark 120 –– – – 92 0 0.74 0.2 Italy Brera et al. (2011)
Dark 20 16 0 0.91 0.23 20 0 0.65 0.39 Canada Turcotte et al. (2013)
Milk 39 –– – – 36 0 0.41 0.08 Germany Engel (2000)
Milk 47 –– – – 47 –– 0.12
b
Spain Burdaspal and Legarda (2003)
Milk 122 –– – – 122 –– 0.10
b
Various Burdaspal and Legarda (2003)
Milk 228 –– – – 228 2 –0.16 Europe Gilmour and Lindblom (2008)
Milk 25 18 0 0.32 0.08 25 0 0.45 0.15 Brazil Copetti et al. (2012b)
Milk 78 –– – – 21 0 0.26 0.15 Italy Brera et al. (2011)
Milk 10 7 0 0.53 0.15 10 0 0.33 0.19 Canada Turcotte et al. (2013)
White 5 –– – – 5–– 0.03
b
Spain Burdaspal and Legarda (2003)
White 9 –– – – 8–– 0.03
b
Various Burdaspal and Legarda (2003)
White 25 5 0 0.1 0.01 23 0 0.05 0.03 Brazil Copetti et al. (2012b)
a
Data not available.
b
Median.
18 M.V. Copetti et al. / International Journal of Food Microbiology 178 (2014) 13–20
Author's personal copy
recommended keeping the cocoa plantation as free of mould infection as
possible, to separate out diseased pods in the fieldanddiscardmummi-
fied pods. The healthy pods should be harvested as soon they are ripe,
avoiding damage to prevent inoculation by fungal spores. Damaged
pods should not be stored longer than one day before opening and
fermenting.
Experiments carried out in Brazil (Copetti et al., 2012a)demonstrat-
ed the importance of organic acids, especially acetic acid, produced by
fermentative bacteria in suppressing the growth of fungi with the po-
tential to produce ochratoxin A, highlighting the importance of an ade-
quate fermentation step. They also showed that fermentation that
occurred during drying of partially depulped beans can increase ochra-
toxin A production, so, a traditional fermentation of 4–7daysshouldbe
adopted, and the mass should be turned frequently. Fermentation be-
yond 7 days should be avoided as this could lead to fungal proliferation
(Codex Alimentarius Commission, 2013).
As toxigenic fungi can grow as fermentation ceases, drying should
start immediately (Copetti et al., 2011b). The code of practice (Codex
Alimentarius Commission, 2013) recommends that layers of drying
cocoa beans should not exceed 6 cm thick to avoid slow or inadequate
drying, and beans should be dried to a moisture content of 6–8%. The
drying area should be located away from contaminant sources and re-
ceive maximum sun exposure and air circulation during the day. At
night or during rainy weather, the cocoa beans should be heaped and
covered to avoid re-wetting.
Dried cocoa beans are hygroscopic, so cocoa will absorb moisture
from the environment under high humidity conditions. Wood (1985)
recommended a maximum 2–3 months storage in tropical countries.
If storage is longer term, humidity should be controlled below 70% RH.
The moisture content of the stored cocoa beans should be periodically
checked and kept below 8% (Codex Alimentarius Commission, 2013).
The formation and reduction of ochratoxin A and aflatoxins during
processing of cocoa beans to cocoa powder and chocolate are shown dia-
grammatically in Fig. 3.Thefigure is purely qualitative, as insufficient data
exists to quantify most stages in formation and reduction. Aflatoxins and
ochratoxin A are shown on the same graph, as qualitative effects are sim-
ilar for both toxins, though quantitative effects are known to vary quite
widely.
7. Final considerations
The concentrations of aflatoxins and ochratoxin A found in cocoa,
cocoa derivatives and chocolate indicate that these products are responsi-
ble for a relatively low contribution to human exposure. However, the in-
crease of chocolate consumption with high levels of cocoa in recent years
could elevate exposure to these food contaminants, as products with a
high cocoa content tend to have the higher concentrations of mycotoxins.
Chocolate appears to be a minor source of ochratoxin A and afla-
toxins in the diet, although the fact that products containing chocolate
are widely consumed by children is a concern, so monitoring of their oc-
currence in these products is important.
Knowledge of factors influencing contamination of cocoa by myco-
toxins has increased in recent years, as well as the processing steps
which may result in a decrease. However, further studies are needed,
particularly on the influence of fermenting microorganisms and the or-
ganic acids they produced on the subsequent inhibition of fungal
growth and mycotoxin production.
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