ARTICLE IN PRESS

Food Control xxx (2003) xxxxxx

www.elsevier.com/locate/foodcont

The production of ochratoxin A by Aspergillus ochraceus in raw

coee at dierent equilibrium relative humidity and under
alternating temperatures
H
ector Palacios-Cabrera a, Marta H. Taniwaki a,*, Hilary C. Menezes b,
Beatriz T. Iamanaka a
a

Instituto de Tecnologia de Alimentos (ITAL), Av. Brasil, 2880, P.O. Box 139, CEP 13073-001, Campinas-SP, Brazil
b
Faculdade de Engenharia de Alimentos (FEA), Unicamp, Campinas-SP, Brazil
Received 26 May 2003; received in revised form 18 August 2003; accepted 19 August 2003

Abstract
The eect of alternating temperatures in the storage of coee was studied. From the day and night values, two average temperatures (25 and 14
C) were chosen. Such changes may occur, mainly during storage in farm barns and transport. The study was
carried out under dierent conditions of equilibrium relative humidity (ERH): 80%, 87% and 95% for the production of ochratoxin
A (OTA) by Aspergillus ochraceus in raw coee. Temperatures were cycled at 12 h intervals. Coee was also maintained at 25
C
under similar conditions, but without temperature cycling, at the same three values of relative humidity. Ochratoxin production was
analysed after periods of 39 and 60 days after the coee had reached the equilibrium relative humidity. The water activity and
moisture content of coee were checked and OTA production was quantied.
There was little or no OTA production at 80% ERH; at 87% and 95% OTA production was high after dierent days of incubation. Under alternating temperatures OTA production was higher than at constant temperature, and alternating temperatures
indirectly favoured OTA production due to condensation and a subsequent rapid increase in moisture content and water activity of
the coee beans.
 2003 Published by Elsevier Ltd.
Keywords: Coee; Ochratoxin A; Aspergillus ochraceus; Alternating temperatures; Water activity

1. Introduction
Coee producing and exporting countries aim to
obtain a good quality safe product and maintain this
throughout the productive chain. However, some factors are out of the control of both producers and exporters. These factors can be climatic or as a result of
logistic structures possibly causing undesirable changes
in the grain characteristics permitting the growth of
certain fungal species and subsequent mycotoxin production.
Of the ochratoxin A (OTA) producing Aspergillus
species, the following were found and isolated from various soils around coee farms: Aspergillus ochraceus,

Corresponding author. Tel.: +55-19 37431819; fax: +55-19

37431822.
E-mail address: mtaniwak@ital.sp.gov.br (M.H. Taniwaki).
0956-7135/$ - see front matter  2003 Published by Elsevier Ltd.
doi:10.1016/j.foodcont.2003.08.006

Aspergillus carbonarius and Aspergillus niger (Joosten,

Goetz, Pittet, Schellenberg, & Bucheli, 2001; Taniwaki,
Pitt, Teixeira, & Iamanaka, 2003; Urbano, Taniwaki,
Leit~ao, & Vicentini, 2001). The presence of OTA in coffee, has been reported by several authors (Kononenko,
Burkin, Zotova, & Soboleva, 2000; Stegen et al., 1997;
Taniwaki et al., 2003; Trucksess, Giler, Young, White, &
Page, 1999).
The inuence of environmental conditions such as
moisture content, temperature, incubation time and the
nature of the substrate, may play an important role in
the colonization by fungi and the amount of OTA
production in dierent grains.
Coee is harvested at dierent moisture contents,
varying from 50% to 70% in ripe cherries, 35% to 50% in
coee raisins (almost dried) and 16% to 30% in the
cherries which dried on the plant (Kamau, 1980). At the
end of the drying process the moisture content must be
at or below 12% to prevent fermentation and mould

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2

H. Palacios-Cabrera et al. / Food Control xxx (2003) xxxxxx

growth (Illy & Viani, 1996). It was shown in previous

studies that the presence of OTA in coee was normally
as a result of badly controlled harvesting procedures,
precarious drying and inadequate storage conditions,
allowing the proliferation of toxigenic fungi (Bucheli,
Meyer, Vuataz, & Viani, 1998; Taniwaki et al., 2003;
Urbano et al., 2001). However, the eect of re-wetting
coee after it has been dried is little known although
there is the possibility that this could also be a cause of
ochratoxin production, especially during storage and
transport.
Drastic changes in temperature may cause metabolic
stress in fungi, which may result in higher or lower toxin
production. Ryu, Munimbazi, and Bullerman (1999)
reported a positive incidence from temperature cycling
in fumonisin production by Fusarium moniliforme and
Fusarium proliferatum.
In the present study, the objective was to articially
increase the moisture gain in dried raw coee contaminated with A. ochraceus, to verify the inuence of alternating temperatures (14 and 25
C) at dierent values
of equilibrium relative humidity (80%, 87% and 95%) on
OTA production, and subsequently determine the
moisture content A. ochraceus needs to produce OTA in
coee under the dierent conditions studied. These
temperatures were chosen from annual data provided by
coee cooperatives in the state of S~
ao Paulo, representing the average values of the main variations in
temperature occurring day and night during coee
storage. Two methods were compared in the determination of the moisture content of the coee: the ISO
method, which has been used as a standard method in
raw coee by several authors (Bucheli et al., 1998;
Gough, 1975), and the vacuum oven method.

2. Material and methods

and transferred into a test tube containing 40 ml phosphate buer plus 0.1% Tween 80 and glass beads, and
the suspension agitated in a vortex for 1 min. A spore
concentration of 107 CFU/ml was obtained by dilution
technique.
2.3. Inoculation of Aspergillus ochraceus spores into the
coee
According to the methodology proposed by PalaciosCabrera et al. (2001), a 1 ml aliquot of spore suspension
(107 CFU/ml) was inoculated into 10 g sterile soil with a
particle size of 300 lm. Subsequently, 4 g of this mixture
were added to 100 g of coee. The coee plus inoculum
was mixed in a Wagner mixer for 15 min. This procedure was repeated for a total weight of contaminated
coee of 1.5 kg.
2.4. Determination of water activity in the raw coee
beans
The water activity (aw ) was measured with the
equipment Aqualab Cx2 (Decagon, USA).
2.5. Calibration curve for the moisture content
The initial moisture content of the raw coee was
determined according to the vacuum oven method at 70

C for 24 h. The Norm International ISO method 1447
(1978) was also used, drying the coee at 130
C for 6 h,
maintaining it in a desiccator at room temperature for
15 h and then drying again at 130
C for a further 4 h.
The calibration curve was prepared in the range from
9% to 20% of coee moisture content.
2.6. Preparation of the saturated salt solutions for use in
the desiccators, obtaining three dierent equilibrium
relative humidity (ERH) values

2.1. Coee
Raw arabica coee was obtained from the experimental farm of the Campinas Agronomy Institute (IAC,
Campinas, SP, Brazil), dried and de-husked. The beans
were sterilised by irradiation using Cobalt 60 at an intensity of 10 kGy at the Agricultural Centre for Nuclear
Energy (CENA/USP, Piracicaba, SP, Brazil).
2.2. Selection and preparation of the Aspergillus ochraceus spore suspensions
Three strains of A. ochraceus CCT 6891 (Tropical
Culture Collection, Fundac~
ao Tropical Andre Tosello,
Campinas, Brazil) were isolated from coee grown in
Brazil. The strains were inoculated into malt extract
agar (MEA) and incubated at 25
C for 5 days. The
growing cultures were mixed to form a single inoculum

Saturated solutions of ammonium sulphate

((NH4 )2 SO4 ), sodium potassium tartrate (NaKC4 H4 O6 )
and lead nitrate (Pb(NO3 )2 ) were prepared, obtaining
solutions at ERH values of 80%, 87% and 95%, respectively. The solutions were placed in the desiccators
and left for 7 days to attain equilibrium.
2.7. Preparation of the isotherm trials at alternating
temperatures of 25 and 14
C and at a constant temperature of 25
C at three dierent values for equilibrium
relative humidity (80%, 87% and 95%)
Plastic pots containing 25 g of dried coee beans were
inoculated with a soil carried spore suspension, as
mentioned before in item 2.3. Five pots were placed in
each desiccator (four contaminated samples and one
non-inoculated control). The desiccators were alter-

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H. Palacios-Cabrera et al. / Food Control xxx (2003) xxxxxx

Moisture content of coffee (dry weight basis)

25

y = 1.0492x + 4.5083
2

R = 0.9974

20

ISO Method

nately placed in chambers at 25 and 14
C (12 h at each

temperature). The ERH values tested were 80%, 87%
and 95%, obtained using the saturated salt solutions.
The non-inoculated pot was weighed at weekly intervals
to determine the increase in moisture and the time needed for the coee beans to reach equilibrium with the salt
solution, based on the change in weight of the beans.
After the beans reached the ERH of the salt solutions,
OTA production was quantied after 39, 46, 53 and 60
days. All trials were carried out in triplicate.
With the aim of obtaining results showing the eect
of temperature alternations on ochratoxin production,
another trial was carried out in parallel under the same
conditions but at a constant temperature of 25
C. In
this trial, the amount of ochratoxin was only quantied
after 39 and 60 days.

15
10
5
0
0

10

15

20

Vacuum oven method

Fig. 1. Calibration curve for raw coee moisture content using two
methodologies (ISO and vacuum oven).

2.8. Extraction and quantication of ochratoxin A in raw

coee beans

3. Results
The calibration curve of the coee moisture content
using two methods (ISO and vacuum oven) is presented
in Fig. 1.
The adsorption isotherm of the raw coee at alternating temperatures of 14 and 25
C is shown in Fig. 2.
The relationship between moisture content and water
activity presented in Fig. 2 allows us to obtain the aw
values of the coee beans from the moisture content
data and vice-versa.
The production of OTA in the beans after reaching
ERH values of 80%, 87% and 95% respectively, at alternating temperatures (every 12 h) of 14 and 25
C, is
presented in Table 1.

Moisture content (dry weight basis)

Adsorption isotherm of raw coffee

OTA was extracted by the method of Pittet, Tornare,

Hugget, and Viani (1996). The coee beans were extracted with a solution of methanol: 3% sodium bicarbonate (50:50). The extracts were ltered and diluted
with phosphate buered saline and applied to an immunoanity column (Vicam, Watertown, MA.) containing a monoclonal antibody specic for OTA. After
washing, the OTA was eluted with HPLC grade methanol and quantied by reverse-phase HPLC using a
uorescence detector. The mobile phase used was
acetonitrile/4 mM sodium acetate with 0.5% acetic acid
solution (42:58). The ow rate was 1 ml/min. The
equipment used was a Shimadzu LC-10VP system
(Shimadzu Corporation, Japan), using a uorescence
detector set at 330 nm excitation and 470 nm emission.
The HPLC was tted with an ODS Hypersil (5 lm, 25
mm 4.6 mm) pre-column and SupelcosilTM LC-18 (5
lm, 250 mm 4.6 mm) column (Supelco, USA). The
detection limit of this method was 0.1 lg/kg OA.

35
2

y = 353.85x - 474.98x + 167.45

2
R = 0.9935

30
25

ISO method

vaccum oven
method

20

y = 369.13x - 495.2x + 179.03

2
R = 0.9936

15
10
5
0
0.65

0.7

0.75

0.8

0.85

0.9

0.95

Water activity (a w)

Fig. 2. Adsorption isotherm of raw coee with cycling temperatures of

14 and 25
C.

Table 1
Production of ochratoxin A (lg/kg) by Aspergillus ochraceus in raw
coee beans at alternating temperatures of 14 and 25
C at dierent
time periods after reaching the equilibrium relative humidity (ERH)
Production of ochratoxin A (lg/kg)a

ERH
(%)

39 days

46 days

53 days

60 days

Un-inoculated
control

80
87
95

NDb
794
2307

ND
1547
6793

1.90
1816
8200

0.35
3869
8338

ND
ND
6.70

a
b

Average of three repetitions.

Not detected.

Under the system of alternating temperatures, the

coee beans took 39 days to reach equilibrium. This
time was established when the weight of the samples
showed no further change to the third decimal place.
The average values determined for the water activity of
the coee beans were: 0.80, 0.86 and 0.90 for 80%, 87%

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H. Palacios-Cabrera et al. / Food Control xxx (2003) xxxxxx

and 95% of ERH, respectively. At 95% ERH, the samples were visibly mouldy.
According to the results in Table 1, it is evident that
aw is a limiting factor for OTA production. At 80%
ERH, there was no OTA production during the rst 46
days, and after 60 days there was only slight production.
The purpose of this study was to reproduce the conditions for OTA production during storage or transport,
when the coee beans had been previously dried and
subsequently stored under high humidity conditions
with changes in day and night temperatures. If the
fungal spores are already present in the coee beans,
OTA production will occur slowly at rst until the coee
reaches a critical equilibrium relative humidity, when at
this point the toxin production will increase sharply. The
amount of ochratoxin found in non-inoculated samples
at 95% ERH must be discounted from the values found
in the samples, but does not signicantly alter the result.
Table 2 presents the data for ochratoxin production
by A. ochraceus in coee maintained at a constant
temperature of 25
C under the same conditions of ERH
(80%, 87% and 95%) used in the previous trial.
At a constant temperature of 25
C, there was no
condensation and the moisture gain was slower. Consequently, the time required to reach equilibrium was
longer than 39 days. The average values for the aw of the
coee beans were: 0.78, 0.84 and 0.88 at 80%, 87% and
95% ERH, respectively. However, to standardize the
data, the analyses were carried out at the same time
intervals of 39 and 60 days as used in the alternating
temperature trial. At 80% ERH, there was little OTA
production as compared to that at 87% and 95% at the
constant temperature of 25
C. At 95% ERH, a water
activity of 0.95 was not reached and the samples were
already mouldy.
Analysing Tables 1 and 2 it can be observed that the
alternating temperature indirectly inuenced the water
activity value. This may be due to condensation, which
took place at the bean surface with the alternating
temperatures. The tendency of this condensation is to
reach equilibrium with the environment. However, the
time the wet coee beans take to equilibrate to their
corresponding water activity could allow for the fungal
Table 2
Production of ochratoxin A (lg/kg) by A. ochraceus in raw coee
beans at a constant temperature of 25
C, after reaching the equilibrium relative humidity (ERH)
ERH (%)

80
87
95
a
b

Production of ochratoxin A (lg/kg)a

39 days

60 days

Un-inoculated
control

NDb
166
2016

0.15
2500
7178

ND
ND
6.58

Average of 3 repetitions.
Not detected.

growth and toxin production to be higher than that

occurring under constant temperature conditions. The
results suggest that it is the water activity rather than the
alternating temperature that is responsible for the higher
OTA production as compared to that at constant temperature.

4. Discussion
In the determination of the moisture content, the ISO
method gave results approximately 4% higher than
those obtained with the vacuum oven method. This
suggests that since the vacuum oven treatment is less
harsh than that of the ISO method, it does not extract
the monolayer water content. According to Gough
(1975) and Lomauro, Bakshi, and Labuza (1985), the
monolayer of absorbed water content of raw coee
corresponds to approximately 4%, which coincides with
the dierence found in this experiment.
The production of OTA by A. ochraceus at ERH
values of 80% was not signicant in any of the trials.
However above 85% ochratoxin production was significant. This is an interesting result, since in a real coee
transport situation there is always a risk of an increase
in moisture content, as a consequence of condensation,
mainly on the grains which are close to the top and/or
the walls of containers. Sharp (1988), Milton and Pawsey (1988) and Pixton (1982) veried this condensation
in maritime routes when there were drastic temperature
changes, mainly when the transport of grains goes from
a warm region to a cold region (sweating container).
One of the reasons why the samples at 80% and 87%
of ERH did not reach the corresponding water activity
(0.80 and 0.87) may be due to the occurrence of condensation resulting from the alternating temperatures of
14 and 25
C. The changes in temperature could lead to
a constant change in adsorption and de-sorption. The
possibility of developing a dierent moisture gradient in
coee stored at alternating temperatures and constant
temperature should also be considered.
These experiments showed the marked inuence of
water activity and incubation time on the production of
OTA in raw coee. They also suggest that fungal growth
could be prevented by an adequate control of environmental factors such as temperature and water activity
and the interaction between them, which are important
in the development of barriers to prevent fungal deterioration in grains (Marin et al., 1998).
Furthermore, these trials conrmed that raw coee is
an adequate substrate for the growth of A. ochraceus
and production of OTA. Mantle and Chow (2000) also
reported high OTA production by A. ochraceus in coffee. Similar work has reported high ochratoxin production (40016 000 lg/kg) in grains such as wheat, rye
and barley, showing that some isolates of A. ochraceus

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H. Palacios-Cabrera et al. / Food Control xxx (2003) xxxxxx

are high producers of this toxin (Chelkowski, Dopierala,

Godlewska, Radomyska, & Szebiotko, 1981; Cvetni
e &
Pepejnjak, 1990; Kononenko et al., 2000).
Northolt and Bullerman (1982) showed that the
minimum aw for the growth of A. ochraceus was between
0.79 and 0.83. In this study, at values of aw between 0.78
and 0.80, there was very little OTA production. However, at 25
C with an aw of 0.84 and at an aw of 0.86
with alternating temperatures of 14 and 25
C, OTA
production increased.
It thus becomes evident that the presence of OTA in
coee is always an indication of serious failures in the
practices of bean harvesting and storage.

Acknowledgements
The authors wish to thank the Cons

orcio Brasileiro
de Pesquisa e Desenvolvimento do Caf
e and Fundac~ao
de Amparo 
a Pesquisa do Estado de S~
ao Paulo (FAPESP) for their nancial support.

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