Effect of operating conditions on Ochratoxin A extraction in roasted

Effect of operating conditions on Ochratoxin A
extraction in roasted coffee
Pauline Mounjouenpou, Noël Durand, Bernard Guyot, Joseph Guiraud
To cite this version:
Pauline Mounjouenpou, Noël Durand, Bernard Guyot, Joseph Guiraud. Effect of operating conditions
on Ochratoxin A extraction in roasted coffee. Food Additives and Contaminants, 2007, 24 (07), pp.730734. <10.1080/02652030701199972>. <hal-00577542>
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Food Additives and Contaminants
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Effect of operating conditions on Ochratoxin A extraction in
roasted coffee
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Journal:
Manuscript Type:
Complete List of Authors:
TFAC-2006-282.R1
Original Research Paper
21-Dec-2006
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Date Submitted by the
Author:
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Manuscript ID:
Food Additives and Contaminants
Methods/Techniques:
Additives/Contaminants:
Chromatography - HPLC, Extraction
Ochratoxin A
Coffee
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Food Types:
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Mounjouenpou, Pauline; Institut de Recherche Agricole pour le
Développement
Durand, Noël; CIRAD-CP, UR33
Guyot, Bernard; CIRAD-CP, UR33
Guiraud, Joseph; Université Montpellier 2, UMR IR2B
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Effect of operating conditions on ochratoxin A extraction
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from roasted coffee
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PAULINE MOUNJOUENPOU1, NOËL DURAND2, BERNARD GUYOT2,
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AND JOSEPH P. GUIRAUD3*
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Institut de Recherche Agricole pour le Développement, BP 2067, Yaoundé, Cameroon1,
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Unité de Recherche 33, CIRAD-CP TA80/16, 34398 Montpellier Cedex 5, France2, and
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UMR-IR2B (Agro-M/INRA/UM2), Université Montpellier II, Place Eugène Bataillon, 34095
Cedex 5, France3.
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Running title: OTA Extraction in Roasted Coffee
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* Author to whom correspondence should be addressed : Joseph-Pierre Guiraud; e-mail :
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guiraud@univ-montp2.fr
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Abstract
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Operating conditions affect ochratoxin A (OTA) extraction from roasted coffee. The OTA
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content found in the beverage can thus be greater than that found in the roasted coffee used to
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prepare it. Three extraction parameters were studied for roasted coffee : type of extraction
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solvent (alkaline, neutral, acid), temperature (ambient temperature/23°C, 60°C and 85°C) and
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extraction time (5, 20, 30, 40, 50, 60 and 80 min). The alkaline solvent that is used in the
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method recommended by the European Union, extracted OTA better, but a maximum content
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was obtained at 60°C after 50 min. At least a 100% improvement in extraction was obtained
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when compared to the EU usual quantification method that is carried out at ambient
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temperature. It turned out that the OTA extraction parameters for roasted coffee, as defined by
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that method, were not optimum and needed to be modified. These results were verified in
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double extraction experiments showing that OTA is not completely extracted by this method.
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Confirmation was obtained by comparison of extraction methods on several commercial
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samples of roasted coffee.
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Keywords
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Roasted coffee; OTA extraction; ochratoxin A
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Introduction
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Ochratoxin A (OTA) is a mycotoxin produced by moulds of the genera Aspergillus and
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Penicillium. In coffee, the OTA-producing strains most frequently found are Aspergillus
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niger, Aspergillus carbonarius and Aspergillus ochraceus (Frank 2001; Joosten et al. 2001;
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Suàrez-Quiroz et al. 2004). OTA has nephrotoxic, immunotoxic, teratogenic and carcinogenic
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effects (Höhler et al. 1998; Pfohl-Leszkowicz and Castegnaro 1999). Its existence in coffee
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was reported for the first time by Levi et al. (1974). Since then, a great deal of work has been
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undertaken to study what happens to the toxin during technological processing of coffee, such
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as roasting and beverage preparation (Viani 1996; Blanc et al. 1998; Van der Stegen et al.
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2001). Some work has indicated a higher OTA content in the beverage (prepared hot)
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compared to that found in the roasted coffee used for beverage preparation (Tsubouchi et al.
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1987; Studer-Rohr et al. 1995; Suàrez-Quiroz et al. 2005,). As the usual method used to
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quantify OTA in roasted coffee (European Union method prEN 14132: 2002 E) recommends
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extraction at ambient temperature, it was important to study how temperature and duration
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influenced OTA extraction. The purpose of this work was therefore to describe the effect of
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different factors (pH, temperature, time) that may affect the OTA extraction in roasted coffee.
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Material and Methods
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Coffee
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The experiments were performed on samples of commercially available roasted, ground
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coffee.
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OTA extraction and quantification parameters
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Extraction was carried out in an alkaline solvent (pH 9,4) consisting of methanol and sodium
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bicarbonate at 3% (20/80; v/v), in a neutral solvent (pH ≈ 6,5) consisting of methanol and
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distilled water (20/80; v/v) and in an acid solvent (pH 4,3) consisting of methanol and
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Titrinorm pH4 buffer (20/80; v/v) (VWR Prolabo, Fontenay sous Bois, France). Extraction
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was carried out either at ambient temperature (23°C), or in a moderately (60°C) or more
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heated medium (85°C), with extraction times of 5, 20, 30, 40, 50, 60 and 80 min. Extraction
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at 85°C required the use of a refrigerant to limit losses through evaporation, and pumice
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stones for stirring during boiling.
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The basic OTA quantification method used was that published by Pittet et al. (1996) for
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soluble coffee, an adaptation of the method developed by Nakajima et al. (1990). The sample
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of roasted coffee (10 g) was extracted at ambient temperature for 30 min with 100 ml of
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alkaline solvent. For that study and all the extraction variants, the filtered extract (5 ml) was
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diluted in 40 ml of alkaline phosphate buffer (PBS) and the mixture was purified on an
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immuno-affinity column (Ochraprep, Rhône Diagnostics, Glasgow, UK). The toxin was
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eluted from the column with 3 ml of methanol and the eluant was evaporated till dry in a
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stream of nitrogen at 70°C. The dry extract was re-suspended in 1 ml of mobile phase
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consisting of distilled water, acetonitrile and acetic acid (51/48/1; v/v/v). Quantification was
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carried out by HPLC (Shimadzu LC-10ADVP, Japan) with fluorimetric detection. The
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operating conditions were as follows:
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column, ODS particle size 5 µm (Lichrospher 50DS2, Interchim, Montluçon, France) with
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identical precolumn, thermostatically controlled at 35°C, isocratic flow of 1ml/min, excitation
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wavelength of 333 nm and emission wavelength of 460 nm. The contents were calculated
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from a calibration curve established from a standard (1 000 ng ml-1 ; ref PD 226 R. Biopharm
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100 µl injection loop, C18 reverse phase HPLC
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Rhône Ltd, Glasgow, UK).
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Each extraction and quantification was performed respectively two and three times and the
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results presented in the form of mean and standard deviation. The limit of quantification of
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the method is 0.03 µg kg-1.
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Results and discussion
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Extraction in an alkaline medium
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Figure 1 shows the curves for OTA extraction in an alkaline medium, depending on the
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temperature and extraction time, for a first batch of coffee. The extraction method
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recommended by the European Union was carried out at ambient temperature for 30 min.
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OTA quantification by that method gave a content equal to 10.5 ± 0.3 µg kg-1. At the same
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temperature, other extraction times gave no change in the amount of OTA quantified.
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However, when extraction was carried out at 60°C, it was clearly improved and then
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depended on the extraction time. The extraction curve passed through a maximum
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(31.0 ± 0.3 µg kg-1) at 50 min. A decrease then set in, which was may be linked to OTA
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degradation or more probably complexing of OTA. Compared to extraction at ambient
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temperature, a maximum extraction improvement of 195% was obtained. At 85°C, OTA
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content decreased immediately and continually in line with the heating time. That decrease
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was a sign of OTA degradation or complexation, which was immediate and probably became
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preponderant compared to the gain in extraction.
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Extraction in a neutral medium
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The curves for OTA extraction in a neutral medium depending on the temperature and
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extraction time are shown in Figure 2 for the same batch of coffee. As in the alkaline medium,
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OTA extraction in a neutral medium at 23°C gave results that remained almost constant,
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irrespective of the extraction time, and which were almost identical (10.3 ± 0.5 µg kg-1), the
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difference in extraction falling within the sensitivity limit of the method (0.03 µg kg-1).
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Consequently, at ambient temperature, there was no significant difference depending on
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whether extraction was carried out in an alkaline or neutral medium. At 60°C, we found a
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gradual and continual rise in OTA content in line with the extraction time. After 80 min, the
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content extracted was 24.4 ± 0.2 µg kg-1. The extraction improvement was therefore 137%
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compared to ambient temperature, but the amount extracted was smaller than that obtained in
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an alkaline medium. At 85°C, the quantities extracted increased in line with the extraction
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time, up to 50 min. At that extraction maximum, the OTA content was 23.1 ± 0.1 µg kg-1,
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which was greater than for extraction at ambient temperature, but was still below the
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maximum obtained in an alkaline medium.
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Extraction in an acid medium
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Figure 3 gives the curves for OTA extraction in an acid medium for the same batch of coffee.
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Extraction at ambient temperature gave quite constant values with a maximum value of 6.4 ±
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1.1 µg kg-1. Heating improved the quantities extracted, but the values remained below those
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obtained with the alkaline and neutral solvents under the same conditions.
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OTA extraction in a neutral or acid medium did not therefore show any particular
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improvement compared with that obtained in an alkaline medium. The best extraction rate
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was obtained with the alkaline solvent at 60°C after 50 min. In order to confirm the fact that
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extraction at ambient temperature was incomplete, a double extraction assay was performed
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on a second batch of coffee.
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Double extraction
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The residue resulting from OTA extraction by the usual method (ambient temperature, i.e.
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23°C for 30 min) was recovered and re-quantified under optimum conditions, i.e. at 60°C for
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50 min. The OTA content found for the first extraction was 16.5 ± 0.2 µg kg-1 (higher content
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than for the first batch of coffee), whereas that found in the residue after the second extraction
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was 9.1 ± 0.1 µg kg-1, i.e. a total of 25.6 µg kg-1. Direct extraction at 60°C gave 26.1 ± 0.6 µg
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kg-1 for the same batch. These results show that OTA extraction conditions at 23°C were not
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optimum and largely underestimated the OTA content. Both the temperature and the
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extraction time were inadequate.
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Verification of the method on several samples of roasted coffee
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Twenty-four different brands of roasted, ground coffee were bought commercially and
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quantified using the method recommended by the EU (alkaline solvent at 23°C for 30 min)
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and the alternative method proposed (alkaline solvent at 60°C for 50 min). Table 1 gives the
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results of a comparison between the two extraction methods on samples of roasted coffee.
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An improvement in OTA extraction, ranging from 100 to 200%, was obtained with the new
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method for all the coffee samples contaminated with OTA. The degree of improvement in the
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extraction rate obtained with the new method varied from sample to sample. That variation
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could be explained by the type of coffee (arabica, robusta, or blend) and the degree of roasting
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(light, medium, dark). During roasting, reactions leading to reversible chelation of OTA with
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other molecules such as proteins can occur (Il’ichev et al. 2002). Figure 4 shows a
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chromatograms of a commercial sample of roasted coffee, a same spiked sample (with 2, 5 µg
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kg-1 added), and a sample of coffee beverage.
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The purpose of this study was to explain the increase in OTA content found in a coffee
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beverage compared to that in the roasted coffee used to prepare it. The increase was due to the
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operating conditions used. The solvent (pH), temperature and extraction time affected the
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OTA content obtained. The EU method (alkaline solvent at ambient temperature for 30 min)
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gave unsatisfactory OTA extraction rates. Alkaline solvent was best for extracting OTA from
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roasted coffee, provided it was extracted at 60°C for 50 min. An improvement of at least
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100% was observed. The results of OTA analysis in roasted coffee or the beverage by the
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conventional technique therefore need to be treated with caution. As the legislation fixes a
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tolerable limit of 5 µg kg-1, it would be advisable to revise the analysis conditions.
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Acknowledgements
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Thanks to the Service for Cooperation and Cultural Action at the French Embassy in
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Cameroon for funding this work.
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References
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Anonymous 2005. Determination of ochratoxin A in barley and roasted coffee - HPLC
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method with immunoaffinity column clean-up. Ref N° prEN 14132:2002 E. CEN B-1050
171
Brussels.
172
Blanc M, Pittet A, Munoz-Boz R, Viani R. 1998. Behaviour of ochratoxin A during green
173
coffee roasting and soluble coffee manufacture. Journal of Agricultural and Food Chemistry
174
46: 673-675.
175
Frank JM. 2001. On the activity of fungi in coffee in relation to Ochratoxin A production. 19th
176
ASIC Coffee Conference, Trieste, Italy. 14-18 May 2001. Available : http://www.asic-
177
cafe.org/htm/eng/proceedings.htm.
178
Höhler D. 1998. Ochratoxin A in food and feed: occurrence, legislation and mode of action.
179
Zeitschrift für Ernäh rungswissenschaft 37: 2-12.
180
Il’ichev YV, Perry JL, Rüker F, Dockal M, Simon JD. 2002. Interaction of Ochratoxin A with
181
human serum albumin. Binding sites localized by competitive interactions with the native
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protein and its recombinant fragments. Chemico-Biological Interactions 141: 275-293.
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Food Additives and Contaminants
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Food Additives and Contaminants
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Joosten HM, Goetz J, Pittet A, Schellenberg M, Bucheli P. 2001. Production of Ochratoxin A
184
by Aspergillus carbonarius on coffee cherries. International Journal of Food Microbiology
185
65: 39-44.
186
Levi CP, Trenk HL, Mohr HK. 1974. Study of the occurrence of Ochratoxin A in green coffee
187
beans. AOAC Journal 57: 866-870.
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Nakajima M, Terada H, Hisada K, Tsubouchi H, Yamamoto K, Uda T, Itoh Y, Kawamura O,
189
Ueno Y. 1990. Determination of Ochratoxin A in coffee beans and coffee products by
190
monoclonal antibody affinity chromatography. Food and Agricultural Immunology 2: 189-
191
195.
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Pfohl-Leszkowicz A, Castegnaro M. 1999. L’Ochratoxine A. In: Les mycotoxines dans
193
l’alimentation: Evolution et gestion des risques. Pfohl-Leszkowicz A, editor. Lavoisier, Paris.
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pp. 249-277.
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Pittet A, Tonare D, Huggett A, Viani R. 1996. Liquid chromatographic determination of
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Ochratoxin A in pure adulterated soluble coffee using an immuno-affinity column cleanup
197
procedure. Journal of Agricultural and Food Chemistry 44: 3564-3569.
198
Studer-Rohr I, Dietrich DR, Schlatter J, Schlatter C. 1995. The occurrence of Ochratoxin A in
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coffee. Food Chemistry and Toxicology 33: 341-355.
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Suàrez-Quiroz ML, Gonzáles-Rios O, Barel M, Guyot B, Schorr-Galindo S, Guiraud JP.
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2004. Study of Ochratoxin A producing strains in coffee processing. International Journal of
202
Food Science and Technology 39: 501-507.
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Suàrez-Quiroz ML, De Louise B, Gonzáles-Rios O, Barel M, Guyot B, Schorr-Galindo S,
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Guiraud JP. 2005. The impact of roasting on the Ochratoxin A content of coffee. International
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Journal of Food Science and Technology 40: 605-611.
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Tsubouchi H, Yamamoto K, Hisada K, Sakabe Y, Udagawa S. 1987. Effect of roasting on
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Ochratoxin A level in green coffee beans inoculated with Aspergillus ochraceus.
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Mycopathologia 97: 111-115.
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Van der Stegen GHD, Essens PJM, Van der Lijn J. 2001. Effect of roasting conditions on
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reduction of Ochratoxin A in coffee. Journal of Agricultural and Food Chemistry 49: 4713-
211
4715.
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Viani R. 1996. Fate of Ochratoxin A (OTA) during processing of coffee. Food Additives and
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Contaminants 13 (suppl.): 29-33.
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Figure captions
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Figure 1. Effect of temperature and extraction time on OTA extraction in an alkaline solvent
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Figure 2. Effect of temperature and extraction time on OTA extraction in a neutral solvent
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Figure 3. Effect of temperature and extraction time on OTA extraction in an acid solvent
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Figure 4. HPLC chromatograms of : commercial sample of roasted coffee, spiked sample with
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2.5 µg OTA kg-1 added, coffee beverage sample
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Page 13 of 16
ee
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Effect of temperature and extraction time on OTA extraction in an alkaline solvent
126x71mm (300 x 300 DPI)
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Food Additives and Contaminants
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Food Additives and Contaminants
ee
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Effect of temperature and extraction time on OTA extraction in a neutral solvent
126x72mm (300 x 300 DPI)
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Page 15 of 16
rR
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Effect of temperature and extraction time on OTA extraction in an acid solvent
127x74mm (300 x 300 DPI)
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Food Additives and Contaminants
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Food Additives and Contaminants
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HPLC chromatograms of commercial sample of roasted coffee, spiked sample with 2.5 µg OTA kg-1
added, coffee beverage sample
239x140mm (96 x 96 DPI)
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