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Stability of ochratoxin A (OTA) during processing and

decaffeination in commercial roasted coffee beans
a

E. A. Nehad , M. M. Farag , M. S. Kawther , A. K. M. Abdel-Samed & K. Naguib

Department of Food Toxicology & Contaminants , National Research Center-Dokki , Dokki,

Cairo, Egypt
b

Biochemistry Department , Faculty of Agricultural Cairo University , Dokki, Cairo, Egypt

Department of Food Toxicology & Contaminants , National Research Center-Dokki , Dokki,

Cairo, Egypt E-mail:
Published online: 20 Feb 2007.

To cite this article: E. A. Nehad , M. M. Farag , M. S. Kawther , A. K. M. Abdel-Samed & K. Naguib (2005) Stability
of ochratoxin A (OTA) during processing and decaffeination in commercial roasted coffee beans, Food Additives &
Contaminants, 22:8, 761-767, DOI: 10.1080/02652030500136852
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Food Additives and Contaminants, August 2005; 22(8): 761767

Stability of ochratoxin A (OTA) during processing and decaffeination in

commercial roasted coffee beans
E. A. NEHAD1, M. M. FARAG2, M. S. KAWTHER1, A. K. M. ABDEL-SAMED2,
& K. NAGUIB1
1

Department of Food Toxicology & Contaminants, National Research Center-Dokki, Dokki, Cairo, Egypt and
Biochemistry Department, Faculty of Agricultural Cairo University, Dokki, Cairo, Egypt

Downloaded by [Korea University] at 21:11 03 January 2015

(Received 4 November 2004; revised 6 April 2005; accepted 8 April 2005)

Abstract
The fate of ochratoxin A (OTA) during the processing of artificially contaminated green coffee beans, the effect
of decaffeination on the production of OTA in green and roasted coffee beans, and the effect of caffeine on the growth
and OTA production by Aspergillus ochraceus were studied. The data indicated that the roasting, milling and decoction
(brewing and Turkish coffee making) processes caused different percentage reductions in OTA. Decaffeinated samples
showed a significantly higher concentration of OTA production than the caffeinated ones. A significantly higher percentage
of OTA was reduced when the decaffeination process was performed before roasting treatment. Caffeine at 1.0 and 2.0%
concentrations completely prevented OTA production and completely inhibited A. ochraceus growth in YES medium after
321 days.

Keywords: Coffee, processing, decaffeination, ochratoxin A

Introduction
The natural occurrence of ochratoxin A (OTA) in
green coffee beans has been reported by several
authors in concentrations ranging between 0.2 and
360 mg kg 1 (Levi et al. 1974; Levi 1980; Norton
et al. 1982; Cantafora et al. 1983; Studer-Rohr et al.
1995). Generally, the removal or reduction of
mycotoxins in food has been demonstrated by several
groups of workers (Scott 1984). In the case of
green coffee beans, the process involved roasting
green coffee. Before that time, it was generally
thought that OTA decomposed during roasting
(Levi et al. 1974; Gallaz and Stalder 1976;
Cantafora et al. 1983). Since then, more experiments
about the fate of OTA during the roasting of coffee
have been published (Tsubouchi et al. 1987; Micco
et al. 1989; Studer-Rohr et al. 1995; Viani 1996;
Blanc et al. 1998; Wilkens et al. 1999; Stegen et al.
2001). In these studies, the results varied from
almost none to almost a complete destruction of
OTA during roasting. However, considerable inconsistencies are found in the literature regarding
the influence of roasting and subsequent operations
on the OTA content of coffee (Viani 1996).

The discrepancy between these findings may be

due to the differences in the method of introducing
the toxin into the ground beans (Tsubouchi et al.
1987) or the fact that the analytical methods
employed until recently would not have been
sufficiently sensitive and selective to detect low
levels of OTA in roasted coffee beans and coffee
decoctions (Pittet et al. 1996). On the other hand, it
was also reported by Viani that these discrepancies
could not be explained either by the different mode
of contamination or by the sensitivity of analytical
method, which can now easily detect levels of OTA
as low as 0.1 mg kg 1. Moreover, three different
explanations were given for this reduction: Physical
removal of OTA with chaff (Blanc et al. 1998),
isomerization at the C3 position into a less toxic
diastereomer (Studer-Rohr et al. 1995) and the
thermal degradation with possible involvement of
moisture (Bourda et al. 1995; Studer-Rohr et al.
1995; Blanc et al. 1998; Stegen et al. 2001).
Regarding the brewing and/or decoction process,
again there is some contradiction among published results. In this concern, Micco et al.
(1989) found no OTA in a brew prepared
from artificially contaminated samples, but both

Correspondence: K. Naguib. E-mail: naguib_kh@hotmail.com

ISSN 0265203X print/ISSN 14645122 online 2005 Taylor & Francis Group Ltd
DOI: 10.1080/02652030500136852

Downloaded by [Korea University] at 21:11 03 January 2015

762

E. A. Nehad et al.

Tsubouchi et al. (1984, 1987) and Studer-Rohr

et al. (1995) measured practically no losses. In this
regard, Stegen et al. (1997) added that OTA present
in roasted coffee essentially passes completely
into the brew with the predominately used brewing methods: Mocha brewing, espresso and
Scandinavian boiled coffee. Moreover, the effect of
decaffeination ought not to be neglected. A 60%
reduction in OTA was measured by Micco et al.
(1989) after industrial decaffeination of naturally
contaminated samples. The inhibitory effects of
caffeine have been reported on the growth of
mycotoxigenic Aspergillus species (Buchanan et al.
1981) and their production of mycotoxins such
as ochratoxin A and aflatoxins (Levi et al. 1974,
1980; Tsubouchi et al. 1984). On the other hand,
caffeine-resistant
strains
of
OTA-producing
A. ochraceus grow well and produce high levels of
OTA in green coffee beans (Tsubouchi et al. 1984).
The objective of the present investigation is to
study the fate of processing and decaffeination on
the stability of OTA in coffee beans. The paper also
deals with the effect of various concentrations
(0.12%) of caffeine on the growth A. ochrouceus
and/or ochratoxin A production in yeast extract
sucrose liquid medium (YES).
Materials and methods
Materials
Ochratoxin A standard. The standard of ochratoxin A
was purchased from Sigma, Chemical Co. (St Louis,
MO, USA).
Ochratoxin A-producing strains. Aspergillus ochraceus.
NRRL 3174 was obtained from the Standard
Association of Australia (North Sydney, NSW,
Australia). The culture was grown on potato
dextrose agar slants at 28 C and stored at 5 C.
Ochratest immunoaffinity column. The ochratest
immunoaffinity columns were purchased from
Vicam (Somerville, MA, USA).
Media. Potato dextrose agar (PDA) (ATCC 1984)
and yeast extract sucrose medium (YES, broth)
(Davis et al. 1969) were used.
Methods
Preparation of a spore suspension of A. ochraceus.
Cultures were regenerated on PDA slants for 7 days
at 2528 C before use. The conidia were harvested
from PDA slants with 5 ml sterilized water and
diluted with buffer containing 0.5% Tween 80.

The final suspension was adjusted to contain approximately 106 conidia ml 1. Each 50 ml YES medium
was inoculated with 1 ml spore suspension and then
incubated for 14 days at 2528 C. After incubation,
the cultures filtrate were analysed for OTA.

Preparation of ochratoxin A standard solution. A stock

solution of OTA was prepared according to the
AOAC Official Method (2000) at a concentration
of 10 mg ml 1 in benzene:acetic acid (99:1 v/v).
The stock solution was stored at 20 C.

Determination of OTA. OTA was determined using

the immunoaffinity column clean-up procedure of
Pittet et al. (1996) and column eluates were
subjected to HPLC analysis. HPLC analysis was
carried out with a liquid chromatograph equipped
with solvent delivery systems (Shimadzu, Columbia,
MD, USA) and a reverse-phase analytical column
packed with C18 material (Spherisorb 5 mm ODS2,
15 cm  4.6 nm). The mobile phase consisted of
acetonitrile:water:acetic acid (99:99:2). Separation
was performed at ambient temperature at a flow
rate of 1.0 ml min 1; the injection volume was 20 ml
for both standard solutions and sample extracts.
The fluorescence detector was operated at an
excitation wave length of 333 nm and an emission
wave length of 460 nm according to the AOAC
(2000).

Preparation of OTA-contaminated samples. Green

coffee beans (100 g) were placed in a 500 ml
Erlenmeyer flask and soaked with 300 ml distilled
water for 56 h. The beans were then decanted and
autoclaved for 10 min at 110 C to sterilize them.
They were inoculated with 1 ml spore suspension
of A. ochraceus and incubated for 30 days at 28 C in
the dark and shaken every day for 10 min to
distribute the mycelia. After incubation the flasks
were autoclaved for 10 min at 110 C. The beans
were then dried for 12 h at 80 C. The infected green
coffee beans were used to study the effect of
processing steps on OTA levels.

Roasting procedure. An infected sample of green

coffee beans was roasted at 180 C for 10 min
(depending on the sample size) in a normal coffee
roaster using a direct flame. The roasted coffee
was ground in high-speed coffee mill (Moulinex)
and passed over a series of manual sieves. A sample
of either infected fine-ground roasted coffee
beans or ground coffee (each 200 g) was analysed
for OTA.

Stability of OTA in commercial roasted coffee beans

Preparation of the coffee brew. Infected roasted
ground coffee (30 g) was placed in a commercial
paper coffee filter and 500 ml boiling water were
poured through the filter. After cooling, 300 ml
coffee brew were taken for OTA analysis.

Statistical Analysis System (SAS Institute 1982).

The significance of the differences among treatment
groups was determined by the WallerDuncan
K-ratio (Waller and Duncan 1969). All statements
of significance were based on probability of p < 0.05.

Preparation of the Turkish coffee. Turkish coffee was

prepared by mixing 30 g infected roasted ground
coffee with 500 ml water. The mixture was heated
at 85 C for 1.5 min. It was then filtered through
Whatman No. 4 filter paper and taken for OTA
analysis.

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763

Results and discussion

Fate of ochratoxin A during processing of green
coffee beans
While the best way to control for the presence of
mycotoxins in food and feed is to prevent their
formation, considerable research has been undertaken on detoxification processes. These might be
intentional treatments of commodities containing mycotoxins that may occur during normal
food and food processing.
The effect of processing on the OTA level
in inoculated green coffee beans is shown in
Table I. The average level of OTA production by
A. ochraceus in the green coffee beans used was
29.36  0.816 mg g 1 after incubation for 30 days
at 2528 C. After roasting treatments at 180 C
for 10 min, the average level of OTA was
20.23  0.816 mg g 1. Roasting thus caused only
a 31.09% destruction of the toxin during green
coffee beans processing. Three different explanations
are given for this reduction: The partial isomerization
of OTA at the C-3 position into a less toxic
diastereomer, thermal degradation and the physical
removal of OTA with chaff (Studer-Rohr et al. 1995;
Blanc et al. 1998; Stegen et al. 2001). Similarly,
Studer-Rohr et al. showed that pure dry OTA is
thermostable and only isomerizes slowly. This was
emphasized by the findings of Bourda et al. (1995)
who showed substantial effects of the presence of
moisture on the reduction of OTA when heating
contaminated wheat.
However, earlier findings showed higher destruction of OTA toxin for such process than the current
results. In this regard, Levi et al. (1974) and Gallaz
and Stalder (1976) found that experimental roasting
operation (20 min at 200  5 C) destroyed about
8090% of the OTA toxin. Moreover, Cantafora
et al. (1983) and Micco et al. (1989) found no
residues of OTA after roasting four green coffee
samples naturally contaminated with 3.8, 23.0 and
4.0, 8.6 ppb OTA, respectively. This is not so

Preparation of decaffeinated coffee. A 1-kg portion of

green coffee was decaffeinated by boiling the beans
in 1 litre water for 5 h, changing the water hourly.
The decaffeinated beans were then dried at 120 C
for 34 h.
Caffeine analysis. The caffeine was determined in
the ground green coffee according to Terada and
Sakabe (1984).
Effect of caffeine on A. ochraceus growth and
OTA production
Ochratoxin A production. A. ochraceus strains (1 ml
spore suspension) were grown in Erlenmeyer flasks
containing 50 ml yeast extract agar medium (YES
broth) supplemented with caffeine at concentrations of 0, 0.1, 0.5, 1.0 and 2.0% (w/v). Then
the flasks were incubated for 3, 6, 9, 12, 15, 18 and
21 days at 25 C. After incubation, the cultures
filtrates were extracted according to Tsubouchi et al.
(1985) and OTA was determined.
Determination of fungal growth. The mycelia were
separated from the YES culture broth by filtration
using Whatman No. 4 filter paper, washing twice
with about 100 ml distilled water, drying overnight
at 100 C and being allowed to cool in a desiccator.
They were then weighed.
Statistical analysis
All data were subjected to statistical analysis using
the General Linear Medial Procedure of the

Table I. Effect of processing steps on ochratoxin A levels in infected green coffee beans.
Initial level
1

Means  SE (mg kg )
Reduction (%)

29.36  0.816

Roasted coffee

Milling coffee

20.23  0.816
31.09

Means superscript with different letters are significantly different ( p < 0.05).
Each value is the average of triplicate samples.

16.09  0.816
45.19

Brewing coffee
d

8.36  0.816
71.52

Turkish coffee
3.67  0.816e
87.5

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764

E. A. Nehad et al.

surprising since the OTA concentrations were

relatively lower in the green beans to an extent that
they can be completely destroyed by the roasting
process (Pittet et al. 1996). The differences between
our data and those reported earlier can be probably
attributed to the difference in the method of
introducing the toxin into the green coffee beans
and/or to the concentration of toxin added. In the
present study, the toxin was produced by inoculating whole beans with toxigenic A. ochraceus. The
toxin produced in this manner may be more strongly
heat resistant as a result of the binding of OTA
to components of the beans and may represent
more accurately the natural contamination of coffee
beans (Pittet et al. 1996). Moreover, the role of
moisture could also explain the wide variation of
OTA reduction at roasting, as observed in the other
studies applying quite unusual roasting conditions,
such as the application of pre-drying or heating
at relatively low temperatures (Stegen et al. 2001).
On the other hand, the obtained results agree with
those given by Studer-Rohr et al. (1995) who found
less than a 30% reduction of OTA toxin in green
coffee beans inoculated with A. ochraceus.
The presented data also revealed that the grinding step has been found to be successful in reducing the level of OTA in the roasted coffee beans. The
average level of OTA detected after grounding the
roasted coffee beans at high speed (Moulinex coffee
mill) and passing them over a series of manual sieves
was 16.09  0.816 mg g 1. This caused a 45.19%
elimination of the toxin, which is mainly due to the
chaff and/or husk removal (Blanc et al. 1998). This
can be explained by coffee husks being more
susceptible to mould growth and OTA production
than the coffee beans themselves. In this concern,
coffee and/or any food adulterated by coffee bean
husks is only an economic issue but may also have an
influence on the safety of the incriminated products.
The same conclusion was reached by Pittet et al.
(1996).
It is clear from Table I that the brewing method
caused destruction of about 71.52% of the OTA
toxin, while the oriental traditional method (Turkish
coffee) was more effective and caused 87.5%
reduction in OTA toxin. This additional reduction
may possibly be due to a limited hydrolysis of OTA
during the high temperature of coffee solubilization
(Blanc et al. 1998) and coffee decoction as well.
It can thus be summarized that the discrepancy
between the findings of different authors is due to
differences in the methods of introducing the toxin
into the ground beans, the initial level of OTA toxin
and the roasting conditions. Another possible reason
for inconsistencies is that analytical methods
employed until recently would not have been
sufficiently sensitive and selective to detect low

levels of OTA in roasted coffee beans and coffee

decoctions (brewing). A major improvement was
introduced by Nakajima et al. (1990), who were the
first to use immunoaffinity chromatography for
the analysis of OTA in coffee products. Pittet et al.
(1996) then developed the method of Nakajima et al.
by using an immunoaffinity columns clean-up
procedure, which was applied in the current study,
to quantify lower levels of OTA in green and ground
roasted coffee beans.
Effect of decaffeination on the production of OTA in green
and roasted coffee beans
The effect of decaffeination on the production of
OTA in green and roasted coffee beans is summarized in Table II. The average levels of OTA
production by A. ochraceus in the caffeinated green
and/or roasted coffee beans were 29.36  0.816
and 19.76  0.926 mg g 1, respectively. The average
levels of OTA production in the decaffeinated
green and/or roasted coffee beans were 49.76 
0.713 and 30.93  0.516 mg g 1 in this order.
Statistical analysis indicated that the analysed
decaffeinated samples had a significantly higher
(p < 0.05) concentration of OTA than caffeinated
ones. This might be probably because caffeine
plays an important role in preventing the formation
of OTA in coffee and the removal of caffeine during
decaffeination increase the risk of OTA contamination. This demonstrates that decaffeination increases
the ochratoxigenic potential of coffee (Nartowicz et
al. 1979; Buchanan et al. 1981).
Moreover, the current data revealed that significantly less ( p < 0.05) OTA concentration was
produced in decaffeinated roasted beans compared
with decaffeinated green beans. This can be probably
attributed to changes in the carbohydrate content of
the coffee beans during roasting (Nartowicz et al.
1979). Sivetz (1963) reported that the sucrose content of coffee decreases upon roasting from 7 to 0%,
while the reducing sugar content remains relatively

Table II. Effect of decaffeination on ochratoxin A production in

green and roasted coffee beans.

Coffee samples

Ochratoxin A
concentration (mg kg 1)*

Green, caffeinated
Green, decaffeinated
Roasted, caffeinated
Roasted, decaffeinated
Decaffeinated roasted beans

29.36  0.816a
49.75  0.713b
19.76  0.926c
30.93  0.516d
3.35  0.686 (88.6%)**

Means superscript with different letters are significantly different

( p < 0.05).
*Each value represents the mean  SE of triplicate samples.
**Values in parentheses represents the per cent reduction in OTA
production.

Stability of OTA in commercial roasted coffee beans

765

Table III. Growth and production of ochratoxin A by A. ochraceus in infected YES medium containing various concentrations of caffeine.

Downloaded by [Korea University] at 21:11 03 January 2015

Caffeine
(%)

Incubation time
(days)

Mycelial dry
weight (mg)a

Ochratoxin A
production (mg ml 1)a

Ochratoxin
A/mycelium (ng mg 1)

Zero

3
6
9
12
15
18
21

1164  14
1471  2
1905  31
2114  14
1739  13
1662  14
1527  2

0.85  0.816
3.72  0.816
3.68  0.816
3.83  0.816
4.05  0.816
4.94  0.816
5.33  0.816

0.73
2.53
1.93
1.81
2.33
2.97
3.49

0.1

3
6
9
12
15
18
21

1104  15 (5)
1447  203 (2)
1726  14 (9)
2005  14 (5)
1582  14 (9)
1618  2 (3)
1520  14 (0.5)

0.49  0.000
1.54  0.016
3.31  0.053
2.08  0.023
1.72  0.024
2.56  0.228
1.49  0.220

0.5

3
6
9
12
15
18
21

151  118 (87)

916  195 (38)
1273  165 (33)
1238  28 (41)
1448  144 (17)
1558  10 (6)
1369  215 (10)

n.d.b
n.d.
n.d.
n.d.
0.975  0.80 (76)
0.939  0.06 (81)
0.855  0.03 (84)

n.d.
n.d.
n.d.
n.d.
0.67 (71)
0.60 (83)
0.63 (82)

1.0

3
6
9
12
15
18
21

314  10 (73)
484  273 (67)
1063  52 (44)
1146  29 (46)
1324  168 (24)
1339  267 (19)
1204  150 (21)

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.

2.0

3
6
9
12
15
18
21

324  15 (72)
493  65 (66)
474  30 (75)
809  114 (62)
831  64 (52)
883  47 (47)
670  29 (56)

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.

(42)
(59)
(10)
(46)
(58)
(48)
(72)

0.44
1.06
1.91
1.04
1.09
1.58
0.98

(40)
(58)
(26)
(43)
(61)
(47)
(72)

Mean  standard error.

No detectable toxin; the value in parentheses represents the per cent inhibition calculated by (1

constant at approximately 1%. Another possible

explanation for the difference in toxin production
between green and roasted decaffeinated samples
may be probably due to the generation of caffeic acid
during roasting, as the chlorogenic acid in green
beans undergoes hydrolysis forming caffeic acid
(Sivetz 1963, 1973; Swaminathan and Koehler
1974). In the same concept, various investigators
(Mateles and Ayde 1965; Davis et al. 1966, 1967;
Davis and Diener 1968) concluded that maximal
aflatoxin synthesis occurs when there are elevated
levels of carbohydrate and the loss of carbohydrate
during roasting might account for the decrease in
toxin production. The same explanations are agreeable, assuming that the OTA biosynthesis might
probably be similarly affected.
It is clear from Table II that the average level
of OTA production by A. ochraceus was
3.35  0.686 mg g 1 when decaffeination was done

test/control)  100.

before roasting. This proves that this treatment

caused an 88.6% destruction of the OTA toxin.
Micco et al. (1989) found that decaffeination alone
caused a 60% reduction of OTA, which was
further increased up to 100% following roasting.
It can thus be concluded that for extra precaution, the green coffee beans must be subjected to
a process of decaffeination before roasting to assure
the absence of OTA in decaffeinated coffee beans.
Effect of caffeine on the growth and ochratoxin A
production by A. ochraceus
Because of the diversity of toxicological manifestation and the economic losses caused by exposure
to OTA, human health must be protected against this
toxin (Marquardt and Frolich 1992). A. ochraceus
is common in green coffee beans and some strains
have the potential to grow on moist green coffee beans

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766

E. A. Nehad et al.

and produce significant amounts of ochratoxins.

Coffee is one of the two extensively consumed
beverages all over the world along with tea.
The growth and OTA production by A. ochraceus
in YES medium containing various concentrations
of caffeine are shown in Table III. The production
of OTA by A. ochraceus in the absence of caffeine
(control) was initiated after 3 days of incubation and
increased to a maximum of 3.49 ng g 1 after 21 days.
In the case of the medium containing 0.1% caffeine,
the production rate was much lower than those
of the control which was 0.44 and 0.98 ng mg 1
after 3 and 21 days of incubation thus causing a
4072% reduction in OTA production. In the same
trend, lower amounts of OTA were observed in
the presence of 0.5% caffeine after 1521 days
of incubation leading to a 7182% reduction.
Caffeine at 1.0 and 2.0% concentrations completely
prevented toxin production (100% reduction).
Table III also shows the effect of caffeine on
the growth of A. ochraceus in YES medium as
determined by mycelial dry weights. The growth
rate of A. ochraceus was inhibited by 2.0% caffeine
after 321 days of incubation (7256% inhibition).
The presence of 0.5 and 1.0% caffeine gave rise to
a delay in the growth of A. ochraceus during 3 days
of incubation and showed 87 and 73% inhibition,
respectively. However, the growth rate of A.
ochraceus was relatively less inhibited after 621
days; reaching 38 to 10 and 67 to 21% inhibition
in this order. However, after 321 days, the growth
rate was no longer inhibited by 0.1% caffeine
(50.5% inhibition). The general pattern of inhibition observed with A. ochraceus growth as well as
the decrease in OTA production shown in our
study was similar to that given by Tsubouchi et al.
(1985). They reported that caffeine has a greater
inhibitory effect on OTA biosynthesis than that
on growth of A. ochraceus. This might be probably
because caffeine interfered with lipid accumulation
(Buchanan et al. 1981) as some fungal species
such A. parasiticus, A. flavus and A. ochraceus
accumulate lipids as a results of secondary metabolism (Gupta et al. 1970; Shin and Marth 1974).
Assuming that the A. ochraceus tested share similar
metabolic orientation, inhibition of lipid accumulation would account for the growth inhibition
pattern observed in the current study (Buchanan
et al. 1981).

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