International Journal of Food Science and Technology 2004, 39, 501–507 501

Study of ochratoxin A-producing strains in coffee

processing

Mirna Suárez-Quiroz,1 Oscar González-Rios,1 Michel Barel,2 Bernard Guyot,2

Sabine Schorr-Galindo1 & Joseph-Pierre Guiraud1*
1 UMR-IR2B (ENSAM/INRA/UM2), Université Montpellier II, Place Eugène Bataillon, 34095 Cedex 5, France
2 CIRAD-CP, TA80/16 34398 Montpellier Cedex 5, France
(Received 15 July 2003; Accepted in revised form 21 November 2003)

Summary Ochratoxin A (OTA) is the main mycotoxin that has been detected in coffee. The
occurrence of OTA in coffee beans can be because of environmental conditions and/or
processing conditions. Three coffee processes were evaluated (wet, mechanical and dry
processes), at different stages from harvesting to storage, and fungi producing OTA were
enumerated and identified. The frequency of potential OTA-producing fungi and their
ability to produce OTA was also studied. By direct plating, the levels of contamination
found in the coffee processes were 80, 72 and 92%, respectively, for parchment and dry
cherry coffee and 20, 34 and 15% for green coffee. Aspergillus ochraceus isolated from the
three processes accounted for 6.6, 8.3 and 3.3%, and Aspergillus niger for 15, 13 and 25%
of the strains isolated, respectively. The toxigenic potential of five A. ochraceus and two
A. niger strains was tested in FDA medium and coffee medium using the HPLC technique.
There was no difference between the processes studied in terms of isolation and occurrence
of ochratoxigenic fungi.
Keywords Aspergillus niger, Aspergillus ochraceus, coffee mycoflora, fungi, post-harvest process.

and closely related species such as Aspergillus

Introduction
Ochratoxin A is a secondary metabolite produced
by several toxigenic species of Aspergillus and
Penicillium. OTA has been shown to exhibit
nephrotoxic, immunosuppressive, teratogenic and
carcinogenic properties (Höhler, 1998). The Inter-
national Agency for Research on Cancer (IARC)
has classified OTA as a possible human carcinogen
(group 2B), based on sufficient evidence for
carcinogenicity in experimental animal studies
and inadequate evidence in humans (IARC,
1993). OTA frequently occurs as a contaminant
in cereals worldwide and it is the main mycotoxic
contamination reported to be found in coffee (Le-
Bars & Le-Bars, 2000). Aspergillus ochraceus is
thought to be the most important OTA-producing
mould in relation to coffee beans (Pitt, 2000).
Recently, it was suggested that Aspergillus niger
*Correspondent: Fax: +33 467 144292;
e-mail: guiraud@arpb.univ-montp2.fr
doi:10.1111/j.1365-2621.2004.00810.x

2004 Blackwell Publishing Ltd
carbonarius could be OTA producers in coffee
(Bucheli et al., 2000; Frank, 2001; Joosten et al.,
2001).
The presence of OTA in coffee is undesirable
because it may be used as a barrier to trade,
affecting the economies of producing countries.
The European Commission has not yet fixed
maximum levels for OTA in coffee, and has only
given one recommendation, in which a reference
level of 3 lg kg)1 was suggested to EU member
states (Romani et al., 2000).
Nine countries have specific regulations for
OTA. Legislative limits range from 5 to
50 lg kg)1 (Boutrif, 1999). If a maximum limit
for OTA in coffee were to be established, it could
affect international trade for producing countries
which do not control this parameter. Without such
limits (uncontrolled) this could have an impact on
human health in coffee importing countries.
The occurrence of OTA in coffee beans can be
due to both environmental conditions (climate,
502 Ochratoxigenic strains in coffee processing M. Suárez-Quiroz et al.
length of storage and transportation) and process-
ing conditions (wet, mechanical or dry processes)
(Romani et al., 2000). The occurrence and the
formation of OTA in the dry process has been
studied by several authors (Bucheli et al., 1998,
2000; Urbano et al., 2001). OTA was present
before storage, indicating the possibility that
harvesting and post-harvest handling of coffee
cherries could be the critical steps leading to
contamination (Bucheli et al., 1998, 2000). There
is currently little information available on the
presence of OTA-producing moulds in coffee
beans used in the wet and mechanical processes
and the impact of these processes on the produc-
tion or presence of OTA. Published data indicate
that the depulping process would significantly
reduce the risk of OTA contamination during the
subsequent fermentation and drying steps, but
mycological studies of these processes are neces-
sary (Frank, 2001). In order to protect coffee from
OTA formation, there is a real need to identify
moulds able to produce this mycotoxin, and their
relation with the processing method.
This research was done so as to enumerate and
identify fungi colonizing coffee beans and to
determinate the frequency of potential OTA-
producing fungi in wet, dry and mechanical
processes at different stages from harvesting to
storage. Their ability to produce OTA was also
studied.
Materials and methods
Coffee samples
Coffee cherries (Coffea arabica var. bourbon,
typica and catimor) were harvested by manual
picking during the 2000–2001 harvest peaks in a
plantation in the Coatepec area (Xalapa, Mexico).
Coffee processing
Three coffee processing methods, wet, mechanical
and dry, were tested on 40-kg batches of coffee
cherries. The trials were repeated four times. The
wet process consisted of the removal of the outer
skin by a mechanical disk pulper and mucilage by
microbial action (fermentation), washing was by
hand. In the mechanical process, a conic vertical
coffee pulper (Penagos Hnos & CIA LTDA,
International Journal of Food Science and Technology 2004, 39, 501–507
Colombia) was used and the mucilage was
removed mechanically with an ascendant vertical
mucilage remover (Penagos Hnos & CIA LTDA,
Colombia). In the dry process, the entire cherry
was directly dried, immediately after harvest. An
air dryer at 45 C was used for the three methods
(rather than the usual sun drying, to ensure more
uniform drying). The first two processes gave
parchment coffee and the other gave dry cherry
coffee. Parchment and husk were removed by
mechanical hulling.
Sampling
Samples (300 g) for mycological analysis were
collected for each method of coffee processing at
different stages of processing from fresh cherries to
storage (Fig. 1). Samples (300 g) of parchment
coffee stored for 1 year were collected using the
same process.
Mycological analysis
Subsamples of cherries or coffee beans were stirred
for 5 min at room temperature in 0.1% peptone
water (1:10 w/v) and 0.1 mL was spread-plated
onto Dichloran Rose Bengal Chloramphenicol
agar (DRBC) (Samson, 1991). The results were
expressed in CFU g)1. Subsamples (fifty beans) of
parchment coffee, dry cherry or green coffee were
also plated directly onto Petri dishes (ten beans per
plate) containing Dichloran 18% Glycerol agar
(DG18) (Hocking & Pitt, 1980; Guiraud, 1998)
without prior superficial disinfection. The plates
were incubated at 25 C for 5–7 days, the results
being expressed as a percentage of infected beans
(% infection). The moulds detected were isolated
and subcultured on Czapek–Dox agar for identi-
fication purposes. Predominant moulds were iden-
tified according to the identification key for
common food-borne fungi (Samson et al., 1995)
and the strains recognized as A. ochraceus Wil-
helm and A. niger van Tieghem were sent to the
BCCMTM/MULC Culture Collection (Leuven
Catholic University, B-1348 Louvain-la-Neuve,
Belgium) for confirmation of the identity. Isolates
identified as A. ochraceus and A. niger were
counted for each sample, and percentage occur-
rences were calculated as a proportion of total
fungi.

2004 Blackwell Publishing Ltd
 Ochratoxigenic strains in coffee processing M. Suárez-Quiroz et al. 503

Figure 1 Coffee processing tested for ochratoxigenic fungi from southeastern Mexico (*sampling).

OTA production by potential producers OTA analysis in coffee beans

Isolates of A. ochraceus and A. niger were grown
on rice media according to the methodology
recommended by FDA (Tournas et al., 2001).
OTA production was then detected and quantified
by HPLC (Nakajima et al., 1997).
In order to study OTA production in coffee, A.
ochraceus and A. niger were grown on PDA agar,
pH 3.5, at 25 C for 5 days. Spores were suspen-
ded in 0.01% Tween 80 solution and 40 g of
coffee beans, obtained by the wet and mechanical
processes, were inoculated with a sample of
15 mL (43 · 106 conidia mL)1) and incubated at
25 C for 15 days (Mantle & Chow, 2000).
OTA production was determined by HPLC
(see below).
Coffee samples were frozen at )80 C then ground
to pass through a 0.5-mm sieve and analysed for
OTA (Nakajima et al., 1997). The samples were
extracted for 30 min with a solution of methanol/
3% sodium bicarbonate (50:50), the extracts were
filtered and diluted with phosphate-buffered saline
and applied to an immunoaffinity column (Och-
raprep,Rhône Diagnostics, Scotland). OTA was
eluted with 3-mL HPLC grade methanol. The
eluate was evaporated to dryness under a stream
of nitrogen at 70 C and the residue was redis-
solved in 1 mL of HPLC mobile phase and then
quantified by HPLC (Shimadzu LC-10ADVP,
Japan, with a fluorescence detector). The mobile
phase consisted of distilled water/acetonitrile/

2004 Blackwell Publishing Ltd International Journal of Food Science and Technology 2004, 39, 501–507
504 Ochratoxigenic strains in coffee processing M. Suárez-Quiroz et al.

glacial acetic acid (51:48:1). The flow rate was
1 mL min)1. OTA was detected by absorption at
333 nm excitation and 460 nm emission, and a
retention time of 13.3–13.5 min. Standard OTA
curves were established with an ochratoxin stand-
ard (1000 ng mL)1 ; ref PD 226 R. Biopharm
Rhône Ltd, Scotland); the detection limit was
0.075 ng mL)1.
Results
Evolution of mould flora during the processes
Table 1 shows the mould counts in beans collected
from the three processes at different stages. Mould
counts reached maximum values of 4.3 ±
0.9 · 102 CFU g)1 in cherries. There was a qual-
itative uniform distribution of filamentous fungi in
samples from the coffee processes tested. All fungi
genera identified had already been recorded in
coffee bean samples from Brazil (Silva et al., 2000;
Urbano et al., 2001; Batista et al., 2003). No
strains of OTA-producing fungi were isolated
from any samples at this stage, but this may have
been because of the detection limit of the tech-
nique used. Consequently, the use of direct plating
of beans in Petri dishes on DG18 agar for samples
giving a negative result (Table 2) was employed. A
high level of infection by moulds was observed in
all processes after drying (on parchment and dry
cherries). It was found that 80% of the beans from
the wet process, 72% from the mechanical process
and 92% from the dry process, analysed as
described earlier, were contaminated with fungi,
Penicillium, Mucor, Cladosporium and Aspergillus
spp. including well-known potential OTA-produ-
cing fungi (A. ochraceus and A. niger). However,
the percentage of infection in green coffee was
lower than that observed in parchment and in dry
cherry coffee, and no OTA-producing fungi were
found. Table 2 shows the percentage occurrence of
A. ochraceus and A. niger isolated from parch-
ment, dry and green coffee for each process; this
corresponded to the analysis of 200 beans at each
stage of processing. One strain of A. ochraceus was
also isolated from parchment coffee produced by
the wet process and stored for 1 year.
OTA production
The toxigenic potential of the isolated fungal
strains was tested and compared to A. ochraceus

)1 a
Table 1 Total mould count (CFU g ) on DRBC agar and main isolates in coffee beans collected at different stages of
processing

Mould countb
Process Stage of processing (CFU g)1) Main strains isolated

Wet process Cherries 4.3 ± 0.9 · 102 Penicillium citrinum, Mucor hiemalis,
P. glabrum, P. minioluteum
Pulped beans 1.8 ± 1.1 · 102 Penicillium citrinum, P. minioluteum
Fermented beans 1.1 ± 0.8 · 102 Geotrichum candidum
Parchment coffee <50c
Green coffee <50c
Mechanical process Cherries 4.3 ± 0.9 · 102 Penicillium citrinum, Mucor hiemalis,
Penicillium glabrum, Penicillium minioluteum
Pulped beans 1.3 ± 0.7 · 102 Penicillium citrinum
Beans with mechanically 7.5 ± 8.6 · 10 Geotrichum candidum
removed mucilage
Parchment coffee <50c
Green coffee <50c
Dry process Cherries 4.3 ± 0.9 · 102 Penicillium citrinum, Mucor hiemalis,
P. glabrum, P. minioluteum
Dry cherries <50b
Green coffee <50b

a
Spread-plated.
b
Mean of four samplings.
c
Limit of method.

International Journal of Food Science and Technology 2004, 39, 501–507
2004 Blackwell Publishing Ltd
 Ochratoxigenic strains in coffee processing M. Suárez-Quiroz et al. 505

Table 2 Rate of coffee bean infection by moulds and percentage occurrence of Aspergillus species potentially capable of
producing ochratoxin A, from different coffee processes

% occurrence

Process Stage of processing % infectiona A. ochraceus A. niger Other species isolated

Wet process Parchment coffee 80 6.6 15 Penicillium glabrum, Mucor sp.,

Cladosporium sp., Aspergillus fumigatus,
A. candidum, A. tamarii, Botrytis sp., P. italicum
Green coffee 20 0 0 Eurotium sp., Fusarium sp., Aspergillus fumigatus
Mechanical Parchment coffee 72 8.3 13 Mucor sp, Aspergillus fumigatus, Penicillium
process citrinum, P. glabrum, P. granulatum
Green coffee 34 0 0 Eurotium chevalieri, Aspergillus fumigatus,
Cladosporium cladosporoides
Dry process Dry cherry 92 3.3 25 Penicillium glabrum, Mucor sp., Cladosporium sp.,
Penicillium sp.
Green coffee 15 0 0 Penicillium minioluteum, Eurotium sp.

a
Direct plating of 200 beans on DG18 agar.

MULC 39534 BCCMTM/MULC Culture Collec-
tion (Table 3). Of the twelve strains of A. ochraceus
detected, five were chosen for their morphological
differences and tested for their ability to produce
OTA: three strains were positive after culture on
rice and coffee media. Production ranged from 0.3
to 679 lg kg)1 of OTA on rice medium and from 1
to 697 lg kg)1 on coffee beans. From the thirty-
nine strains of A. niger isolated, two strains, chosen
at random, were tested for the production of OTA
in rice and coffee media and all gave positive
results, ranging from 2.2 to 114 lg kg)1 of OTA.
No strains of A. carbonarius and P. verrucosum
were isolated in this study.
Table 4 shows the levels of OTA (lg kg)1)
from samples testing positive for A. ochraceus and
A. niger. For all samples, the analysis was done
after hulling on green coffee, parchment or husk.
The presence of OTA was correlated with the
presence of OTA-producing strains.
Discussion
The mould counts from coffee were generally low
and no well-known potential OTA-producing
fungi could be isolated before drying. Similar
observations were made by several authors
(Frank, 1999; Bucheli et al., 2000), who failed to
isolate ochratoxigenic moulds from fresh cherries;
this was attributed to the presence of species
adapted to high moisture (yeast, bacteria or other
moulds) that may inhibit their growth. However, a

Table 3 Comparison of OTA pro-

duction in different media by iso- OTA production (lg kg)1)
lates of Aspergillus ochraceus and
FDA rice Coffee beana Coffee beanb
Aspergillus niger from different
Isolate Origin medium medium medium
coffee processing methods
A. ochraceus Reference strain 19 1.8 1
A. ochraceus 232 Wet process nd 2.3 1.3
A. ochraceus 243 Wet process nd nd nd
A. ochraceus 237 Mechanical process 0.3 2.6 1.3
A. ochraceus 246 Mechanical process nd nd nd
A. ochraceus 259 Wet process 679 697 188
(1999 harvest)
A. niger 250 Wet process 5.7 6.2 9.8
A. niger 296 Dry process 114 5.1 2.2

a
Beans from mechanical process.
b
Beans from wet process.
nd, not detected.

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506 Ochratoxigenic strains in coffee processing M. Suárez-Quiroz et al.

Table 4 Relationship between levels of OTA contamination produce OTA in the coffee medium, though for
in green coffee, parchment or husk and isolates of one strain of A. ochraceus, OTA production was
Aspergillus ochraceus and Aspergillus niger present in
observed in the coffee medium but not in the FDA
coffee beans
medium: 75 and 3% of A. ochraceus and A. niger
Level of OTA isolates, respectively, were OTA producers
contamination (lg kg)1)b (Taniwaki et al., 2003). Studies of factors influen-
Species of potential
Parchment OTA-producing cing toxigenesis showed that toxin production can
Samplea Green coffee or husk fungi isolated vary by as much as 1000-fold among isolates from
the same species (Le-Bars & Le-Bars, 2000). This
232 0.2 0.3 A. ochraceus
study is in accordance with the observations made
243 nd nd A. ochraceus
237 nd 0.1 A. ochraceus by other authors (Taniwaki et al., 2003), who
246 nd nd A. ochraceus reported that infection by A. ochraceus and A.
259 0.2 Trace A. ochraceus niger did not necessarily indicate high OTA
250 0.2 0.3 A. niger contamination. However, the results obtained in
296 0.2 0.3 A. niger
this study indicated that toxigenesis does not
a
Sample number assigned for laboratory control. appear to be affected by the process to which the
b
HPLC. coffee is subjected.
nd, not detected. When a toxigenic strain was isolated from a
high level of contamination by yeast and moulds, coffee sample, the sample contained OTA. This
including A. ochraceus and A. niger, was observed shows that the presence of toxigenic strains
in raw coffee beans collected in different producing involves a great risk of OTA presence.
regions of Brazil (Urbano et al., 2001). Contrary to what might be expected from the
Independently of the method used, during results in Table 2, it was shown that hulling coffee
processing a substantial decrease in the mould containing OTA did not totally eliminate the toxin,
count was found, which was expressed in an which was subsequently found in green coffee.
apparent absence (limit of detection) of ochra- In this work, it was not possible to monitor
toxigenic moulds in parchment and dry cherry changes in the toxigenic strains right from the first
coffee and later on in green coffee. Using the viable stage (coffee cherries). This work will be comple-
count methodology it was not possible to prove ted by a study based on pilot fermentations with
the existence of ochratoxigenic strains in parch- artificially contaminated coffee cherries.
ment and dry cherry coffee. Nevertheless, by the
direct plating technique, several strains of A. niger Acknowledgments
and A. ochraceus were isolated from beans. It can
be seen that the average rate of parchment and dry We thank Mr C.I. Hernández-Palacios, Mexico,
cherry coffee infection by these fungi was low (3– for providing the coffee samples and processing
25%). This method made it possible to determine facilities, UNIDA of the Veracruz Technology
the percentage of beans contaminated by moulds, Institute and the LATEX Laboratory at Veracruz
which was high in parchment coffee (71–92%) University for laboratory facilities.
decreasing later on in green coffee (15–34%).
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