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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Aflatoxin B1 and ochratoxin A in dried vine fruits from Greek market a
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Eleni Kollia , Alexandros Kanapitsas & Panagiota Markaki
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Department of Food Chemistry,Faculty of Chemistry , National and Kapodistrian University of Athens , Athens , Greece Accepted author version posted online: 29 Jul 2013.
To cite this article: Food Additives & Contaminants: Part B (2013): Aflatoxin B1 and ochratoxin A in dried vine fruits from Greek market, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2013.825647 To link to this article: http://dx.doi.org/10.1080/19393210.2013.825647
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Aflatoxin B1 and Ochratoxin A in dried vine fruits from Greek Market
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Eleni Kollia, Alexandros Kanapitsas, Panagiota Markaki
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Department of Food Chemistry, Faculty of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis Zografou 15784 Athens, Greece
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Twenty-six samples of dried vine fruits from Athens and Thebes (Central Greece) market were simultaneously extracted and cleaned up by immunoaffinity columns and analyzed for AFB1 and OTA. A combination of ELISA and HPLC methods was applied for the determination of AFB 1. Recovery was 97.6 %, RSD 6.46%, while the limit of detection (LOD) and limit of quantification (LOQ) were 0.05 μg kg-1 and 0.09 μg kg-1 respectively. OTA concentrations were only estimated ELISA. Results revealed the presence of AFB1 in 23% of the samples (mean 1.4 μg AFB1 kg -1), but none exceeded the EU limit (2 μg AFB1 kg-1). However, OTA was detected in 100% of the samples (mean 47.2 μg OTA kg-1). Six samples were found to be contaminated at high levels (median 120.6μg OTA kg-1) and 18 exceeded the EU limit (10 μg OTA kg-1).
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Abstract
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Keywords: Aflatoxin B1, Ochratoxin A, HPLC, ELISA, Dried vine fruits.
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Corresponding author: P.Markaki, Department of Food Chemistry, School of Chemistry, University of Athens, Panepistimiopolis Zografou, GR-157 84 Athens, Greece, tel. 0030-210-7274489, Fax 0030-2107274476, email: [email protected]
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Introduction
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The mycotoxins of greatest significance in foods and feeds are aflatoxins (AFs), which are mainly produced by A.flavus and A.parasiticus. Aflatoxins are difuranocoumarin derivatives which are naturally occurring in foodstuff and are likely to contaminate nuts, dried fruits, spices, etc. AFB 1 is regarded to be genotoxic and the most potent liver carcinogen for many animal species as well as for humans and is classified as human carcinogen (group 1) by the International Agency of Research on Cancer (IARC 1993a). In addition the mycotoxin ochratoxin A (OTA) which is produced by Aspergillus ochraceus and related species in agricultural commodities, is a potent nephrotoxic, teratogen and carcinogen. Exposure to OTA has been associated with kidney disease known as BEN (Balkan Endemic Nephropathy) (Pfohl-Leszkowicz et al. 2002). IARC classified OTA as a possible human carcinogen (group 2B) (IARC 1993b). Speijers and Speijers (2004) reported that OTA and AFB1 are among the most frequent observed combinations of mycotoxins in different plant products. According to Bircan (2009) dried vine fruits are commodities which could support aflatoxigenic and ochratoxigenic mold growth as well as AFB1 and OTA production.
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Due to the fact that toxinogenic species of Aspergillus ochraceus, Penicillium verrucosum and Aspergillus carbonarius can grow and produce OTA at moderate, low and high temperatures, respectively, widespread occurrence of OTA has been reported in a variety of different geographical regions and climates, in several foods and beverages (Janati et al. 2012). Moreover, surveys for OTA have been carried out in many countries such as Spain, UK, Germany, including Greece (Battilani et al. 2006). AFB1 was found in dried vine fruits from India (Saxena and Mehrotra 1990) and Egypt (Abdel-Sater and Saber 1999; Youssef et al. 2000). According to Trucksess and Scott (2008) the most frequently reported occurrence of AFs is in dried figs and raisins. Raisins are produced in the United States, Turkey, Greece and Australia. (Fernández-Cruz María et al. 2010; Asadi et al. 2012).
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The European Union (EU) has established limits for mycotoxins in many products at high risk of contamination. As regards AFB1 the maximum level in dried fruits for direct human consumption is 2 μg kg-1, whereas for OTA the maximum level is 10 μg kg-1. The maximum limit of AFB1 in dried vine fruits subjected to sorting or physical treatment is 5μg kg-1 (European commission 2006). The present study reports the occurrence of AFB1 and OTA when they are simultaneously extracted from dried vines fruit purchased from the Greek market. In addition the present study evaluates the efficiency of a routine method for AFB 1 determination in dried vine fruits by HPLC. AFB1 and OTA were also determined by using the method ELISA (Enzyme Linked Immunosorbent Assay).
Material and methods
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Samples
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Twenty six samples of dried vine fruits (currants, raisins and sultanas), both unpacked and packed, conventional and organic were collected randomly from markets of Athens and Thebes (Central Greece) during 2012. The samples were of Greek origin, while three of them were imported from America and Turkey. The conventional dried vine fruits from open containers (n =19) were two currant samples originated from Crete, four raisin samples from Crete, six raisin samples from Corinth, six currant samples from Corinth and one currant sample originated from America. The conventional packaged samples (n= 5) were two sultana samples originated from Turkey, one sultana sample from Aigio (North Peloponnese), one currant sample from Aigio and one currant sample from Corinth. The organic packaged samples (n= 2) were, one currant sample from Corinth and one sultana sample from Crete.
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As regards the retail unpacked dried vine fruits, 1 kilo was purchased, from which 5 incremental samples weighing 100 g each were taken, consequently the laboratory sample was 500 g. Representative subsamples of 150 g from the laboratory sample were minced with a sterilized mincer and mixed well. Portions of 15 g were taken from the homogenate for the determination of AFB1 and OTA. As regards the retail packed dried vine fruits, four retail packs with the same lot number (250 g pack -1) from every kind were purchased and mixed into 1 kg. Two incremental samples weighing 250 g each were taken, consequently the laboratory sample was 500 g. These were processed in the same way. Samples were stored in polyethylene bags and kept at 4 oC until analysis. Before analysis, samples were brought to room temperature. The determination of AFB1 was performed by HPLC and ELISA. The determination of AFB 1 by HPLC was in-house characterized. The determination of Ochratoxin A was only performed by using ELISA.
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Apparatus
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ELISA was performed by a Multi-Mode Microplate Reader (Synergy™ HT, BioTek, Winooski, USA). HPLC was performed using a Hewlett-Packard 1050 (Hewlett-Packard, Waldbron, Germany) Liquid Chromatograph equipped with a JASCO FP-920 (Jasco Ltd., Tokyo, Japan) fluorescence detector and an HP integrator 3395. The HPLC column used was a C18 Nova-Pak (60 Å, 4 μm, 4.6 x 250 mm) (Waters-Millipore, Milford, MA, USA). Mobile phase [water + acetonitrile + methanol (20 + 4 + 3)] for AFB 1 determination was filtered through Millipore HA-VLP (0.45 μm) filters before use. The derivative of AFB1 (AFB2a, the hemiacetal of AFB1) was detected at λex = 365 nm/λem = 425 nm. The flow rate was 1 ml min-1 and the retention time was 15.6 ± 0.7 min.
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Reagents
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AFB1 standard was purchased from Sigma (St. Louis, MO, USA). Aflatest and Ochratest immunoaffinity columns were obtained from VICAM (Waters, Watertown, MA, USA). All reagents used were of analytical grade while HPLC solvents were of HPLC grade and were purchased from Fisher Chemical (Fisher Scientific, Loughborough, Leicestershire, UK). Hexane and methanol (pro analysis) were from Merck (Darmstadt, Germany), trifluoroacetic acid was obtained from Fluka (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), ELISA kits Ridascreen, were from R-Biopharm AG (Darmstadt, Germany). Phosphate-buffered saline (PBS) solution was prepared by dissolving 0.2 g potassium chloride, 0.2 g potassium dihydrogen phosphate, 1.16 g anhydrous disodium hydrogen phosphate and 8.0 g of sodium chloride in 900 mL of distilled water. After adjusting the pH to 7.4 using 0.1 M HCI/0.1 M NaOH if necessary, the solution was made up to 1000 mL.
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Determination of AFB1 A sample of dried vine fruits (15 g) was mixed with 30 mL methanol + water (80 + 20 v/v) and shaken well for 10 min. After filtration an aliquot of 1 mL from the filtrate was used for AFB 1 analysis. Clean-up: 1 mL from the filtrate was diluted with 10 mL of water and mixed for 1 min. The mixture was loaded onto the Aflatest column (flow rate 3 mL/min) and washed twice with 10 mL of water. The column was then allowed to dry by passing air. AFB1 was carefully eluted with 2 mL of acetonitrile (flow rate = 0.3 mL/min). The eluate was divided into two parts of 1 ml. The first part was analyzed by ELISA and the second by HPLC (Daradimos et al. 2000).
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Determination by ELISA
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The first part of eluate was evaporated to dryness under a gentle stream of nitrogen. Thereafter the evaporated solution was redissolved in 400 μl of methanol + water (70:30) for analysis. Then 50 μl of samples and standard solutions were inserted into the wells of microwell holder. Detection was performed by measuring the absorbance at 450 nm and results were calculated by using the standard curve y= - 0.323ln(x) +1.523, r2=0.9932, where y= the absorbance and x= the corresponding concentration of AFB1 in the microwells.
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Determination by HPLC
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The second part of the eluate was evaporated to dryness under a gentle stream of nitrogen. A derivative of AFB 1 (AFB2a, the hemiacetal of AFB1) was prepared by adding 200 μl hexane and 200 μl trifluoroacetic acid to the evaporated solution of AFB1, heated at 40o C in a water bath for 10 min, evaporated to dryness, redissolved in 200 μl of water + acetonitrile (9 + 1) and analyzed by HPLC. AFB2a shows enhanced fluorescence compared to AFB1 (Stubblefield 1987).
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Determination of OTA
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OTA extraction from dried vine fruits is described in the section ‘‘Determination of AFB 1”. For clean-up 5 mL from the filtrate were diluted with 40 mL PBS and mixed for 1 min. The mixture was loaded onto the Ochratest column at a flow rate of 3 mL min-1 and washed once with 20 mL water. The column was then allowed to dry by passing air. OTA was carefully eluted with 2 mL of a solution of methanol + acetic acid (98:2 v/v) at a flow rate of 0.3 mL min-1. The eluate was evaporated to dryness under a gentle stream of nitrogen. The residue was dissolved immediately in 400 μl of water + methanol (70:30 v/v) and analyzed by ELISA. 50 μl of each sample and standard solution was inserted into the wells of the microwell holder. Detection was performed by measuring the absorbance at 450 nm and results were calculated by using the standard curve y= -0.563ln(x) +2.6072, r2=0.993, where y= the absorbance and x= the corresponding concentration of OTA in the microwells.
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Results and Discussion
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Characterization of the AFB1 determination in dried vine fruits
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Analysis of complex food such as dried vine fruits, requires validation of the analytical method (Gilbert and Anklam 2002). In the present study the analytical protocol for the determination of AFB1 in dried vine fruits was characterized in-house for the following criteria: linearity, accuracy, repeatability, internal reproducibility, limits of detection (LOD) and limits of quantification (LOQ). Additionally, repeatability (r) and reproducibility (R) were calculated to be r= 2.8×SDr and R=2.8×SDR respectively. LOD and LOQ were calculated according to LOD=[b0 + 3S(b0)]/b1 and LOQ=[b0 + 10S(b0)]/b1, where b1 is sensitivity (calibration model slope), Sb0 is the standard deviation of the blank and b0 is the response of the blank (intercept of the calibration model).
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Repeatability was estimated by analyzing 4 subsamples (15g) of dried vine fruits spiked with 100 ng AFB1, corresponding to 6.7 μg AFB1 kg-1 under repeatability conditions (mean recovery = 113.5%, RSD= 2.8%). Intra-laboratory reproducibility was estimated by determining 4 sub-samples (15g) of dried vine fruits at time intervals on four different days of the month (mean recovery = 112.2%, RSD= 1.2%). Repeatability (r) and reproducibility (R) were r= 8.96 and R= 3.75, respectively (SD r= 3.20 and SDR= 1.34). Accuracy of the method was studied by analyzing 15 g sub – samples of dried vine fruits spiked with AFB1 at different quantities, as shown in Table 1. The regression data of the curve were found to be r= 0.9932, y= 0.9736 (± 0.0465)x + 0.0638 (± 0.0941), RSD%= 6.46, where y is the concentration of AFB 1 (μg kg-1) of dried vine fruits recovered and x is the concentration of AFB1(μg kg-1) of dried vine fruits spiked.
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Recently Janati et al. (2012) reported an LOD for AFB1 of 0.2 μg kg-1 and recoveries for raisins and currants of 88.9% and 90% respectively by using IAC cleanup and HPLC determination with fluorescence detection. Earlier Imperato et al. (2011) reported a LOD of 0.08 μg kg-1 and an average recovery of 95.4% for AFB1 in dried figs. Compared to these findings the LOD (0.045 μg AFB1 kg-1) and recovery (97.6%) in the present study are very satisfactory.
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Characterization of AFB1 and OTA determination by ELISA
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AFB1 and OTA were also investigated by ELISA, which has the advantage that no cleanup is required. However, drawbacks of the ELISA can be possible cross reactivities of the antibodies, which can lead to false positives (Reiter et al. 2009). In the present study the extraction was not the same as proposed by R-Biopharm but as described in the materials and methods paragraph. Moreover, a cleanup procedure with IAC minicolumns Aflatest and Ochratest was used before the determination by ELISA. In that case the results for OTA, revealed by ELISA test, are not considered as doubtful. LODs for ELISA were 1.6 μg kg -1 and 0.8 μg kg-1 for AFB1 and OTA respectively, following the procedures as described in this study. LODs reported in the manuals were 1 μg AFB1 kg-1 and 5 μg OTA kg-1. AFB1 rates determined with ELISA were below the detection limit of the method. Therefore all samples were then analyzed by HPLC. On the other hand as already mentioned OTA occurred in all samples. Control samples of dried vine fruits spiked with a known concentration of AFB1 (50 ng flask-1, corresponding to 3.3 μg kg-1) and non-spiked samples were processed according to the procedure as described. Determination with ELISA did not demonstrate false positive or false negative results for these control samples.
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Occurrence of AFB1 in dried vine fruits determined by HPLC
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AFB1 was detected in 23 % of the samples examined (mean = 0.15 μg AFB 1 kg-1, median = 0.05 μg AFB1 kg-1), when determined by HPLC. As shown in Table 3, AFB1 was detected in six samples of different varieties of dried vine fruits examined. The concentration of AFB1 in these six samples was below the EU limit (2 μg AFB1 kg-1). Most contaminated was a raisins sample from Corinth (0.6 μg AFB1 kg-1). Moreover one sample of currants from Corinth (conventional, packaged), one sample of sultanas from Aigio (conventional, packaged), one sample of currants from Corinth (organic, packaged) and one sample of sultanas from Crete (organic, packaged) were found to be contaminated with AFB 1 at 0.05μg AFB1 kg-1. One sample of currants originated from Corinth (conventional, open container) and one sample of raisins originated from Corinth (conventional, open container) were found to be contaminated with AFB 1 at 0.1 μg AFB1 kg-1 and 0.6 μg AFB1 kg-1 respectively. Results were not corrected for recovery. Iamanaka et al. (2007) reported AFB1 concentrations in sultanas from Brazil of 2 μg AFB1 kg-1. On the other hand AFB1 in raisins from India were at higher levels (180 μg AFB1 kg-1) (Saxena and Mehrotra 1990). High levels were also found in Egypt: 550 μg AFB1 kg-1 (AbdelSater and Saber 1999) and 300 μg AFB1 kg-1 (Youssef et al. 2000). Lower levels (7.5 μg kg-1) were reported by Luttfullah and Hussain (2011) in dried fruits.
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Occurrence of OTA in dried vine fruits
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A Fisher test was applied to the linear regression data. The F(1, 15) ratio was 86.5, which is above the critical Fisher value of 16.59 at an alpha risk 0.1% for 1 and 5 degrees of freedom. Therefore the regression model can be considered to be acceptable. Finally, the lack of fit of the model was found to be negligible since the experimental F ratio (1,76) is lower than the critical value F (3, 15) = 9.34 with a risk a=0.1% at df= 3,15. Therefore, the field of linearity chosen was approved. The mean recovery factor of the method taken from the above equation was 97.6% (RSD= 6.46%). LOD was determined at 0.27 μg kg-1 and LOQ at 0.82 μg kg-1dried vine fruits. If LOD and LOQ were defined at a signal-to-noise ratio 3:1, they would be 0.045 μg kg-1 and 0.09 μg kg-1, respectively. Throughout this study, the lowest LOD (= 0.045 μg AFB1 kg-1) was taken into account. AFB1 content was below LOD in blank samples. The in-house characterization of the method’s uncertainty was measured considering the lower and the higher AFB1 quantities recovered from samples spiked with 0.3 μg AFB1 kg-1 and 3.3 μg AFB1 kg-1 respectively (Table 1). Uncertainty calculated as 2x RSD% and was 8.2 and 18.6 for the lower and higher spike respectively.
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OTA was revealed in 100% of the dried vine fruits samples examined (n = 26; mean = 47.2 μg OTA kg, median = 21.5 μg OTA kg-1, range from 2.8 to 138.3 μg OTA kg-1). A percentage of 69.2% from all samples were contaminated above the limit of 10 μg kg-1 as established by EU legislation (Commission Regulation (EC) No. 1881/2006). Most contaminated samples were three currant samples from Corinth (138.3 μg OTA kg -1, 123.4 μg OTA kg-1, 101.5 μg OTA kg-1), one raisin sample from Crete (103.3 μg OTA kg-1) as well as currants and sultanas from Aigio (136.3 μg OTA kg-1, 117.8 μg OTA kg-1) (Table 2). These results agree with FernandezCruz et al. (2010), who reported OTA contamination in dried vine fruits, in several countries at maximum levels 1
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In the literature it is reported that OTA is formed during the drying stage. Although OTA levels are low in the drying stage, they might increase during transportation, storage and processing if the conditions are favorable (Karbancıoglu-Güler and Heperkan 2008). This is probably the reason for the high incidence of OTA found in dried vine fruits purchased from the Greek market. Moreover, in the present study six samples of dried vine fruits were concomitantly contaminated by AFB1 and OTA (Table 3.) According to Speijers and Speijers (2004), mycotoxins with similar mode of action could have additional effects. The mode of action for AFB1 and OTA is different. However, Huff et al. (1992) reported that the toxicity to broilers resulting from the combination of these toxins is more severe than when either of these toxins was present alone. In the present study all samples were contaminated with OTA at high levels. On the contrary, AFB 1 contamination was found only in five samples at low levels. Imperato et al. (2011) stated that among food products analyzed in Italy, dried vine fruits were mainly contaminated with OTA and less with AFs. Revealed from the literature, when OTA is present at high levels, AFB1 contamination is low and vice versa. As it is already noticed by MacDonald et al. (1999), no AFB1 was found in dried vine fruits, while OTA was detected at high levels. In addition, it is reported that OTA inhibits AFB1 production by Aspergillus parasiticus (Dimitrokallis et al. 2008).
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ranging from 26 to 250μg kg 1. In addition, high levels of OTA taken from the literature, were 35 μg kg-1 in raisins analyzed in Sweden (Möller & Nyberg 2003), 250 μg kg -1 in Egyptian raisins (Youssef et al. 2000), 26 μg kg-1 in sultanas examined in Canada (Lombaert et al. 2004), 54 μg kg-1 in unprocessed sultanas in Turkey (Meyvaci et al. 2005), 100 μg kg-1 in processed Turkish sultanas (Aksoy et al. 2007) and 54 μg kg-1 in currants analyzed in the UK (MacDonald et al. 1999). Previous surveys reported incidences rates of OTA in raisins of 97% (Ministry of Agriculture, Fish and Foods (MAFF) 1999), while a German survey (Engel 2000) reported a a 95% overall incidence rate of OTA in raisins and currants. Data from Finland and France indicated incidence rates of 71% and 46%, respectively (Miraglia and Brera 2002). In the present study, samples of sultanas and currants taken from the Greek market in 2012, contaminated by OTA, were around 44- and 57-fold higher, in comparison with results reported for sultanas and currants taken from the Greek market in 2003 (Stefanaki et al. 2003). In Greece, the highest frequency of OTA in currants and sultanas occurred in samples from sea level and the lowest occurred in samples from the highest (600–1000m) altitudes (Pateraki 2005).This is in agreement with the present study, since most contaminated samples were currants from North Peloponnese (Corinth and Aigio).
Conclusions
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Although the number of samples examined is limited, the fact that OTA was detected in a percentage of 100% and eighteen samples were found to be highly contaminated (11.4 –138.3 μg kg-1) this indicates the potential of OTA occurrence in dried vine fruits. According to Pardo et al. 2005 inadequate storage conditions allow the proliferation of toxigenic fungi. Prevention is preferable to destruction or removal of the mycotoxins since the latter is not fully efficient (Norholt and Bullerman 1995). On the other hand AFB 1 occurrence was limited and samples were found to be contaminated at low levels. Thus efforts have to be made to prevent mold growth and mycotoxin production along the entire food chain from field to table. To this purpose, control of all factors (temperature, packaging, environmental conditions) during storage would result in producing final products of high quality and safety, inhibiting contamination from toxigenic molds and mycotoxin production.
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Acknowledgement
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The authors wish to thank the Laboratory of Food Microbiology and Biotechnology of Agricultural University of Athens and the director Professor G.J. Nychas (Department of Food Science and Technology) for providing the Multi-Mode Microplate Reader (Synergy™ HT, BioTek, Winooski, USA).
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Miraglia, M., and Brera, C.(2002). Task 3.2.7 ‘Assessment of Dietary Intake of Ochratoxin A by the Population of EU Member States’. Reports on Tasks for Scientific Cooperation, Directorate General Health and Consumer Protection (Brussels: European Commission).
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Molinie, A., Faucet, F., Castegnaro, M., Pfohl-Leszkowicz, A.(2005). Analysis of some breakfast cereals on the French market for their content of ochratoxin A, citrinin and fumonisin B 1: development of a new method for simultaneous extraction of ochratoxin A and citrinin. Food Chem, 92: 391–400
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Möller, T.E., Nyberg, M. (2003). Ochratoxin A in raisins and currants: basic extraction procedure used in two small marketing surveys of the occurrence and control of the heterogeneity of the toxins in samples. Food Addit Contam, 20: 1072–1076.
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Pardo E. Modelling of effects of water activity and temperature on germination and growth of ochratoxigenic isolates of Aspergillus ochraceus on a green coffee-based medium. Int J Food Microbiol, 98(1): 11-9
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Pateraki, M., Dekanea, A., Mitchell, D., Lydakis, D. & Magan, N. (2005). Ecology and control of ochratoxin in grapes and dried vine fruits. BCPC Crop Science and Technology, 5B: 403-410.
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Pfohl-Leszkowicz, A., Petkova-Bocharova, T., Chernozemsky, I.N., Castegnaro M., (2002). Balkan endemic nephropathy and associated urinary tract tumors: a review on etiological causes and the potential role of mycotoxins Food Addit Contam., 19: 282–302
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Reiter, E., Zentek, J., & Razzazi, E. (2009). Review on sample preparation strategies and methods used for the analysis of aflatoxins in food and feed. Mol Nutr Food Res, 53: 508–524
308 309
Saxena, J.,Mehrotra, B.S. (1990). The occurrence of mycotoxins in some dry fruits retail marketed in Nainital district of India. Acta Aliment Hung, 19: 221–224.
310
Speijers, G.J.A., Speijers, M.H.M.(2004). Combined toxic effects of mycotoxins, Toxicol Lett, 153: 91-98.
311 312
Stefanaki, I., Foufa, E., Tsatsou-Dritsa, A., and Dais, P., (2003). Ochratoxin A concentrations in Greek domestic wines and dried vine fruits, Food Addit Contam, 20: 74–83.
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Stubblefield, R.D.(1987). Optimum conditions for formation of aflatoxin M1-trifluoroacetic acid derivative. J Assoc Off Ana Chem,7: 1047-1049.Sweeney, M.J., Dobson, A.D.W. (1998). Mycotoxin production by Aspergillus, Fusarium and Penicillium species. Int J Food Microbiol, 43: 141-158.
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Trucksess M. W. & Scott P. M. (2008). Mycotoxins in botanicals and dried fruits: A review. Food Additives and Contaminants, 25(2): 181–192.
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Youssef, M.S., Abo-Dahab, N.F., Abou-Seidah, A.A. (2000), Mycobiota and mycotoxin contamination of dried raisins in Egypt. Afr J Mycol Biotechnol, 8,69-86, Chemical Abstracts 135:317617r, 2001.
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Meyvaci, K.B., Altindisli, A., Aksoy, U., Eltem, R.,Turgut, H.,Arasiler, Z.,Kartal, N.(2005). Ochratoxin A in sultanas from Turkey I: Survey of unprocessed sultanas from vineyards and packing-houses. Food Addit Contam, 22: 1138–1143.
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320
321
Table 1
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Accuracy of the method for AFB1 determination applied on dried vine fruits 323 324 AFB1 spiked in dried vine fruits (μg kg-1)
325
t
Samples 0.7
1.3
2.7
326 3.3
2.7
3.2
2.7
3.7
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0.3
0.8
1.7
2
0.3
0.7
1.5
3
0.3
0.7
4
0.3
0.6
Mean
0.3
0.7
SD
0
0.1
0
1.3
2.2
3.0
1.3
2.8
3.4
1.5
2.6
3.3
0.2
0.3
0.3
13
10
9
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RSD%
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AFB1 (μg kg-1) recovered
327 328
Table 2
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OTA in samples of dried vine fruits Type and origin
Range (μg OTA kg-1) n
< 0.8-10
10-100
>100
Crete
2
-
2
-
Corinth, Aigio (Peloponnese)
9
1
4
4
America
1
-
1
Crete
3
2
-
Corinth, Aigio (Peloponnese)
6
6
Turkey
2
-
Aigio (Peloponnese)
1
Crete
1
332
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331
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1 -
2
-
-
-
1
1
-
-
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330
-
-
Sultanas
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Raisins
t
Currants
333 334 Table 3 335 AFB1 and OTA co-occurrence in samples of dried vine fruits Concentration OTA (μg kg-1)
0.1
101.5
Currants (Corinth)
0.05
t
AFB1 (μg kg-1)
Currants (Corinth)
Currants (Corinth)
0.05
Sultanas (Aigio)
0.05
Sultanas (Crete)
0.05
Raisins (Corinth)
0.6
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93.5 39.4
117.8
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9.8
an
336
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Samples
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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Aflatoxin B1 and ochratoxin A in dried vine fruits from Greek market a
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Eleni Kollia , Alexandros Kanapitsas & Panagiota Markaki
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Department of Food Chemistry,Faculty of Chemistry , National and Kapodistrian University of Athens , Athens , Greece Accepted author version posted online: 29 Jul 2013.
To cite this article: Food Additives & Contaminants: Part B (2013): Aflatoxin B1 and ochratoxin A in dried vine fruits from Greek market, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2013.825647 To link to this article: http://dx.doi.org/10.1080/19393210.2013.825647
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Aflatoxin B1 and Ochratoxin A in dried vine fruits from Greek Market
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Eleni Kollia, Alexandros Kanapitsas, Panagiota Markaki
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Department of Food Chemistry, Faculty of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis Zografou 15784 Athens, Greece
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Twenty-six samples of dried vine fruits from Athens and Thebes (Central Greece) market were simultaneously extracted and cleaned up by immunoaffinity columns and analyzed for AFB1 and OTA. A combination of ELISA and HPLC methods was applied for the determination of AFB 1. Recovery was 97.6 %, RSD 6.46%, while the limit of detection (LOD) and limit of quantification (LOQ) were 0.05 μg kg-1 and 0.09 μg kg-1 respectively. OTA concentrations were only estimated ELISA. Results revealed the presence of AFB1 in 23% of the samples (mean 1.4 μg AFB1 kg -1), but none exceeded the EU limit (2 μg AFB1 kg-1). However, OTA was detected in 100% of the samples (mean 47.2 μg OTA kg-1). Six samples were found to be contaminated at high levels (median 120.6μg OTA kg-1) and 18 exceeded the EU limit (10 μg OTA kg-1).
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Abstract
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Keywords: Aflatoxin B1, Ochratoxin A, HPLC, ELISA, Dried vine fruits.
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Corresponding author: P.Markaki, Department of Food Chemistry, School of Chemistry, University of Athens, Panepistimiopolis Zografou, GR-157 84 Athens, Greece, tel. 0030-210-7274489, Fax 0030-2107274476, email: [email protected]
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Introduction
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The mycotoxins of greatest significance in foods and feeds are aflatoxins (AFs), which are mainly produced by A.flavus and A.parasiticus. Aflatoxins are difuranocoumarin derivatives which are naturally occurring in foodstuff and are likely to contaminate nuts, dried fruits, spices, etc. AFB 1 is regarded to be genotoxic and the most potent liver carcinogen for many animal species as well as for humans and is classified as human carcinogen (group 1) by the International Agency of Research on Cancer (IARC 1993a). In addition the mycotoxin ochratoxin A (OTA) which is produced by Aspergillus ochraceus and related species in agricultural commodities, is a potent nephrotoxic, teratogen and carcinogen. Exposure to OTA has been associated with kidney disease known as BEN (Balkan Endemic Nephropathy) (Pfohl-Leszkowicz et al. 2002). IARC classified OTA as a possible human carcinogen (group 2B) (IARC 1993b). Speijers and Speijers (2004) reported that OTA and AFB1 are among the most frequent observed combinations of mycotoxins in different plant products. According to Bircan (2009) dried vine fruits are commodities which could support aflatoxigenic and ochratoxigenic mold growth as well as AFB1 and OTA production.
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Due to the fact that toxinogenic species of Aspergillus ochraceus, Penicillium verrucosum and Aspergillus carbonarius can grow and produce OTA at moderate, low and high temperatures, respectively, widespread occurrence of OTA has been reported in a variety of different geographical regions and climates, in several foods and beverages (Janati et al. 2012). Moreover, surveys for OTA have been carried out in many countries such as Spain, UK, Germany, including Greece (Battilani et al. 2006). AFB1 was found in dried vine fruits from India (Saxena and Mehrotra 1990) and Egypt (Abdel-Sater and Saber 1999; Youssef et al. 2000). According to Trucksess and Scott (2008) the most frequently reported occurrence of AFs is in dried figs and raisins. Raisins are produced in the United States, Turkey, Greece and Australia. (Fernández-Cruz María et al. 2010; Asadi et al. 2012).
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The European Union (EU) has established limits for mycotoxins in many products at high risk of contamination. As regards AFB1 the maximum level in dried fruits for direct human consumption is 2 μg kg-1, whereas for OTA the maximum level is 10 μg kg-1. The maximum limit of AFB1 in dried vine fruits subjected to sorting or physical treatment is 5μg kg-1 (European commission 2006). The present study reports the occurrence of AFB1 and OTA when they are simultaneously extracted from dried vines fruit purchased from the Greek market. In addition the present study evaluates the efficiency of a routine method for AFB 1 determination in dried vine fruits by HPLC. AFB1 and OTA were also determined by using the method ELISA (Enzyme Linked Immunosorbent Assay).
Material and methods
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Samples
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Twenty six samples of dried vine fruits (currants, raisins and sultanas), both unpacked and packed, conventional and organic were collected randomly from markets of Athens and Thebes (Central Greece) during 2012. The samples were of Greek origin, while three of them were imported from America and Turkey. The conventional dried vine fruits from open containers (n =19) were two currant samples originated from Crete, four raisin samples from Crete, six raisin samples from Corinth, six currant samples from Corinth and one currant sample originated from America. The conventional packaged samples (n= 5) were two sultana samples originated from Turkey, one sultana sample from Aigio (North Peloponnese), one currant sample from Aigio and one currant sample from Corinth. The organic packaged samples (n= 2) were, one currant sample from Corinth and one sultana sample from Crete.
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As regards the retail unpacked dried vine fruits, 1 kilo was purchased, from which 5 incremental samples weighing 100 g each were taken, consequently the laboratory sample was 500 g. Representative subsamples of 150 g from the laboratory sample were minced with a sterilized mincer and mixed well. Portions of 15 g were taken from the homogenate for the determination of AFB1 and OTA. As regards the retail packed dried vine fruits, four retail packs with the same lot number (250 g pack -1) from every kind were purchased and mixed into 1 kg. Two incremental samples weighing 250 g each were taken, consequently the laboratory sample was 500 g. These were processed in the same way. Samples were stored in polyethylene bags and kept at 4 oC until analysis. Before analysis, samples were brought to room temperature. The determination of AFB1 was performed by HPLC and ELISA. The determination of AFB 1 by HPLC was in-house characterized. The determination of Ochratoxin A was only performed by using ELISA.
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Apparatus
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ELISA was performed by a Multi-Mode Microplate Reader (Synergy™ HT, BioTek, Winooski, USA). HPLC was performed using a Hewlett-Packard 1050 (Hewlett-Packard, Waldbron, Germany) Liquid Chromatograph equipped with a JASCO FP-920 (Jasco Ltd., Tokyo, Japan) fluorescence detector and an HP integrator 3395. The HPLC column used was a C18 Nova-Pak (60 Å, 4 μm, 4.6 x 250 mm) (Waters-Millipore, Milford, MA, USA). Mobile phase [water + acetonitrile + methanol (20 + 4 + 3)] for AFB 1 determination was filtered through Millipore HA-VLP (0.45 μm) filters before use. The derivative of AFB1 (AFB2a, the hemiacetal of AFB1) was detected at λex = 365 nm/λem = 425 nm. The flow rate was 1 ml min-1 and the retention time was 15.6 ± 0.7 min.
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Reagents
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AFB1 standard was purchased from Sigma (St. Louis, MO, USA). Aflatest and Ochratest immunoaffinity columns were obtained from VICAM (Waters, Watertown, MA, USA). All reagents used were of analytical grade while HPLC solvents were of HPLC grade and were purchased from Fisher Chemical (Fisher Scientific, Loughborough, Leicestershire, UK). Hexane and methanol (pro analysis) were from Merck (Darmstadt, Germany), trifluoroacetic acid was obtained from Fluka (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), ELISA kits Ridascreen, were from R-Biopharm AG (Darmstadt, Germany). Phosphate-buffered saline (PBS) solution was prepared by dissolving 0.2 g potassium chloride, 0.2 g potassium dihydrogen phosphate, 1.16 g anhydrous disodium hydrogen phosphate and 8.0 g of sodium chloride in 900 mL of distilled water. After adjusting the pH to 7.4 using 0.1 M HCI/0.1 M NaOH if necessary, the solution was made up to 1000 mL.
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Determination of AFB1 A sample of dried vine fruits (15 g) was mixed with 30 mL methanol + water (80 + 20 v/v) and shaken well for 10 min. After filtration an aliquot of 1 mL from the filtrate was used for AFB 1 analysis. Clean-up: 1 mL from the filtrate was diluted with 10 mL of water and mixed for 1 min. The mixture was loaded onto the Aflatest column (flow rate 3 mL/min) and washed twice with 10 mL of water. The column was then allowed to dry by passing air. AFB1 was carefully eluted with 2 mL of acetonitrile (flow rate = 0.3 mL/min). The eluate was divided into two parts of 1 ml. The first part was analyzed by ELISA and the second by HPLC (Daradimos et al. 2000).
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Determination by ELISA
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The first part of eluate was evaporated to dryness under a gentle stream of nitrogen. Thereafter the evaporated solution was redissolved in 400 μl of methanol + water (70:30) for analysis. Then 50 μl of samples and standard solutions were inserted into the wells of microwell holder. Detection was performed by measuring the absorbance at 450 nm and results were calculated by using the standard curve y= - 0.323ln(x) +1.523, r2=0.9932, where y= the absorbance and x= the corresponding concentration of AFB1 in the microwells.
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Determination by HPLC
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The second part of the eluate was evaporated to dryness under a gentle stream of nitrogen. A derivative of AFB 1 (AFB2a, the hemiacetal of AFB1) was prepared by adding 200 μl hexane and 200 μl trifluoroacetic acid to the evaporated solution of AFB1, heated at 40o C in a water bath for 10 min, evaporated to dryness, redissolved in 200 μl of water + acetonitrile (9 + 1) and analyzed by HPLC. AFB2a shows enhanced fluorescence compared to AFB1 (Stubblefield 1987).
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Determination of OTA
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OTA extraction from dried vine fruits is described in the section ‘‘Determination of AFB 1”. For clean-up 5 mL from the filtrate were diluted with 40 mL PBS and mixed for 1 min. The mixture was loaded onto the Ochratest column at a flow rate of 3 mL min-1 and washed once with 20 mL water. The column was then allowed to dry by passing air. OTA was carefully eluted with 2 mL of a solution of methanol + acetic acid (98:2 v/v) at a flow rate of 0.3 mL min-1. The eluate was evaporated to dryness under a gentle stream of nitrogen. The residue was dissolved immediately in 400 μl of water + methanol (70:30 v/v) and analyzed by ELISA. 50 μl of each sample and standard solution was inserted into the wells of the microwell holder. Detection was performed by measuring the absorbance at 450 nm and results were calculated by using the standard curve y= -0.563ln(x) +2.6072, r2=0.993, where y= the absorbance and x= the corresponding concentration of OTA in the microwells.
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Results and Discussion
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Characterization of the AFB1 determination in dried vine fruits
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Analysis of complex food such as dried vine fruits, requires validation of the analytical method (Gilbert and Anklam 2002). In the present study the analytical protocol for the determination of AFB1 in dried vine fruits was characterized in-house for the following criteria: linearity, accuracy, repeatability, internal reproducibility, limits of detection (LOD) and limits of quantification (LOQ). Additionally, repeatability (r) and reproducibility (R) were calculated to be r= 2.8×SDr and R=2.8×SDR respectively. LOD and LOQ were calculated according to LOD=[b0 + 3S(b0)]/b1 and LOQ=[b0 + 10S(b0)]/b1, where b1 is sensitivity (calibration model slope), Sb0 is the standard deviation of the blank and b0 is the response of the blank (intercept of the calibration model).
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Repeatability was estimated by analyzing 4 subsamples (15g) of dried vine fruits spiked with 100 ng AFB1, corresponding to 6.7 μg AFB1 kg-1 under repeatability conditions (mean recovery = 113.5%, RSD= 2.8%). Intra-laboratory reproducibility was estimated by determining 4 sub-samples (15g) of dried vine fruits at time intervals on four different days of the month (mean recovery = 112.2%, RSD= 1.2%). Repeatability (r) and reproducibility (R) were r= 8.96 and R= 3.75, respectively (SD r= 3.20 and SDR= 1.34). Accuracy of the method was studied by analyzing 15 g sub – samples of dried vine fruits spiked with AFB1 at different quantities, as shown in Table 1. The regression data of the curve were found to be r= 0.9932, y= 0.9736 (± 0.0465)x + 0.0638 (± 0.0941), RSD%= 6.46, where y is the concentration of AFB 1 (μg kg-1) of dried vine fruits recovered and x is the concentration of AFB1(μg kg-1) of dried vine fruits spiked.
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Recently Janati et al. (2012) reported an LOD for AFB1 of 0.2 μg kg-1 and recoveries for raisins and currants of 88.9% and 90% respectively by using IAC cleanup and HPLC determination with fluorescence detection. Earlier Imperato et al. (2011) reported a LOD of 0.08 μg kg-1 and an average recovery of 95.4% for AFB1 in dried figs. Compared to these findings the LOD (0.045 μg AFB1 kg-1) and recovery (97.6%) in the present study are very satisfactory.
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Characterization of AFB1 and OTA determination by ELISA
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AFB1 and OTA were also investigated by ELISA, which has the advantage that no cleanup is required. However, drawbacks of the ELISA can be possible cross reactivities of the antibodies, which can lead to false positives (Reiter et al. 2009). In the present study the extraction was not the same as proposed by R-Biopharm but as described in the materials and methods paragraph. Moreover, a cleanup procedure with IAC minicolumns Aflatest and Ochratest was used before the determination by ELISA. In that case the results for OTA, revealed by ELISA test, are not considered as doubtful. LODs for ELISA were 1.6 μg kg -1 and 0.8 μg kg-1 for AFB1 and OTA respectively, following the procedures as described in this study. LODs reported in the manuals were 1 μg AFB1 kg-1 and 5 μg OTA kg-1. AFB1 rates determined with ELISA were below the detection limit of the method. Therefore all samples were then analyzed by HPLC. On the other hand as already mentioned OTA occurred in all samples. Control samples of dried vine fruits spiked with a known concentration of AFB1 (50 ng flask-1, corresponding to 3.3 μg kg-1) and non-spiked samples were processed according to the procedure as described. Determination with ELISA did not demonstrate false positive or false negative results for these control samples.
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Occurrence of AFB1 in dried vine fruits determined by HPLC
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AFB1 was detected in 23 % of the samples examined (mean = 0.15 μg AFB 1 kg-1, median = 0.05 μg AFB1 kg-1), when determined by HPLC. As shown in Table 3, AFB1 was detected in six samples of different varieties of dried vine fruits examined. The concentration of AFB1 in these six samples was below the EU limit (2 μg AFB1 kg-1). Most contaminated was a raisins sample from Corinth (0.6 μg AFB1 kg-1). Moreover one sample of currants from Corinth (conventional, packaged), one sample of sultanas from Aigio (conventional, packaged), one sample of currants from Corinth (organic, packaged) and one sample of sultanas from Crete (organic, packaged) were found to be contaminated with AFB 1 at 0.05μg AFB1 kg-1. One sample of currants originated from Corinth (conventional, open container) and one sample of raisins originated from Corinth (conventional, open container) were found to be contaminated with AFB 1 at 0.1 μg AFB1 kg-1 and 0.6 μg AFB1 kg-1 respectively. Results were not corrected for recovery. Iamanaka et al. (2007) reported AFB1 concentrations in sultanas from Brazil of 2 μg AFB1 kg-1. On the other hand AFB1 in raisins from India were at higher levels (180 μg AFB1 kg-1) (Saxena and Mehrotra 1990). High levels were also found in Egypt: 550 μg AFB1 kg-1 (AbdelSater and Saber 1999) and 300 μg AFB1 kg-1 (Youssef et al. 2000). Lower levels (7.5 μg kg-1) were reported by Luttfullah and Hussain (2011) in dried fruits.
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Occurrence of OTA in dried vine fruits
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A Fisher test was applied to the linear regression data. The F(1, 15) ratio was 86.5, which is above the critical Fisher value of 16.59 at an alpha risk 0.1% for 1 and 5 degrees of freedom. Therefore the regression model can be considered to be acceptable. Finally, the lack of fit of the model was found to be negligible since the experimental F ratio (1,76) is lower than the critical value F (3, 15) = 9.34 with a risk a=0.1% at df= 3,15. Therefore, the field of linearity chosen was approved. The mean recovery factor of the method taken from the above equation was 97.6% (RSD= 6.46%). LOD was determined at 0.27 μg kg-1 and LOQ at 0.82 μg kg-1dried vine fruits. If LOD and LOQ were defined at a signal-to-noise ratio 3:1, they would be 0.045 μg kg-1 and 0.09 μg kg-1, respectively. Throughout this study, the lowest LOD (= 0.045 μg AFB1 kg-1) was taken into account. AFB1 content was below LOD in blank samples. The in-house characterization of the method’s uncertainty was measured considering the lower and the higher AFB1 quantities recovered from samples spiked with 0.3 μg AFB1 kg-1 and 3.3 μg AFB1 kg-1 respectively (Table 1). Uncertainty calculated as 2x RSD% and was 8.2 and 18.6 for the lower and higher spike respectively.
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OTA was revealed in 100% of the dried vine fruits samples examined (n = 26; mean = 47.2 μg OTA kg, median = 21.5 μg OTA kg-1, range from 2.8 to 138.3 μg OTA kg-1). A percentage of 69.2% from all samples were contaminated above the limit of 10 μg kg-1 as established by EU legislation (Commission Regulation (EC) No. 1881/2006). Most contaminated samples were three currant samples from Corinth (138.3 μg OTA kg -1, 123.4 μg OTA kg-1, 101.5 μg OTA kg-1), one raisin sample from Crete (103.3 μg OTA kg-1) as well as currants and sultanas from Aigio (136.3 μg OTA kg-1, 117.8 μg OTA kg-1) (Table 2). These results agree with FernandezCruz et al. (2010), who reported OTA contamination in dried vine fruits, in several countries at maximum levels 1
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In the literature it is reported that OTA is formed during the drying stage. Although OTA levels are low in the drying stage, they might increase during transportation, storage and processing if the conditions are favorable (Karbancıoglu-Güler and Heperkan 2008). This is probably the reason for the high incidence of OTA found in dried vine fruits purchased from the Greek market. Moreover, in the present study six samples of dried vine fruits were concomitantly contaminated by AFB1 and OTA (Table 3.) According to Speijers and Speijers (2004), mycotoxins with similar mode of action could have additional effects. The mode of action for AFB1 and OTA is different. However, Huff et al. (1992) reported that the toxicity to broilers resulting from the combination of these toxins is more severe than when either of these toxins was present alone. In the present study all samples were contaminated with OTA at high levels. On the contrary, AFB 1 contamination was found only in five samples at low levels. Imperato et al. (2011) stated that among food products analyzed in Italy, dried vine fruits were mainly contaminated with OTA and less with AFs. Revealed from the literature, when OTA is present at high levels, AFB1 contamination is low and vice versa. As it is already noticed by MacDonald et al. (1999), no AFB1 was found in dried vine fruits, while OTA was detected at high levels. In addition, it is reported that OTA inhibits AFB1 production by Aspergillus parasiticus (Dimitrokallis et al. 2008).
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ranging from 26 to 250μg kg 1. In addition, high levels of OTA taken from the literature, were 35 μg kg-1 in raisins analyzed in Sweden (Möller & Nyberg 2003), 250 μg kg -1 in Egyptian raisins (Youssef et al. 2000), 26 μg kg-1 in sultanas examined in Canada (Lombaert et al. 2004), 54 μg kg-1 in unprocessed sultanas in Turkey (Meyvaci et al. 2005), 100 μg kg-1 in processed Turkish sultanas (Aksoy et al. 2007) and 54 μg kg-1 in currants analyzed in the UK (MacDonald et al. 1999). Previous surveys reported incidences rates of OTA in raisins of 97% (Ministry of Agriculture, Fish and Foods (MAFF) 1999), while a German survey (Engel 2000) reported a a 95% overall incidence rate of OTA in raisins and currants. Data from Finland and France indicated incidence rates of 71% and 46%, respectively (Miraglia and Brera 2002). In the present study, samples of sultanas and currants taken from the Greek market in 2012, contaminated by OTA, were around 44- and 57-fold higher, in comparison with results reported for sultanas and currants taken from the Greek market in 2003 (Stefanaki et al. 2003). In Greece, the highest frequency of OTA in currants and sultanas occurred in samples from sea level and the lowest occurred in samples from the highest (600–1000m) altitudes (Pateraki 2005).This is in agreement with the present study, since most contaminated samples were currants from North Peloponnese (Corinth and Aigio).
Conclusions
224 225 226 227 228 229 230 231 232
Although the number of samples examined is limited, the fact that OTA was detected in a percentage of 100% and eighteen samples were found to be highly contaminated (11.4 –138.3 μg kg-1) this indicates the potential of OTA occurrence in dried vine fruits. According to Pardo et al. 2005 inadequate storage conditions allow the proliferation of toxigenic fungi. Prevention is preferable to destruction or removal of the mycotoxins since the latter is not fully efficient (Norholt and Bullerman 1995). On the other hand AFB 1 occurrence was limited and samples were found to be contaminated at low levels. Thus efforts have to be made to prevent mold growth and mycotoxin production along the entire food chain from field to table. To this purpose, control of all factors (temperature, packaging, environmental conditions) during storage would result in producing final products of high quality and safety, inhibiting contamination from toxigenic molds and mycotoxin production.
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Acknowledgement
235 236 237
The authors wish to thank the Laboratory of Food Microbiology and Biotechnology of Agricultural University of Athens and the director Professor G.J. Nychas (Department of Food Science and Technology) for providing the Multi-Mode Microplate Reader (Synergy™ HT, BioTek, Winooski, USA).
238 239
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320
321
Table 1
322
Accuracy of the method for AFB1 determination applied on dried vine fruits 323 324 AFB1 spiked in dried vine fruits (μg kg-1)
325
t
Samples 0.7
1.3
2.7
326 3.3
2.7
3.2
2.7
3.7
cr ip
0.3
0.8
1.7
2
0.3
0.7
1.5
3
0.3
0.7
4
0.3
0.6
Mean
0.3
0.7
SD
0
0.1
0
1.3
2.2
3.0
1.3
2.8
3.4
1.5
2.6
3.3
0.2
0.3
0.3
13
10
9
M
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12
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0.3
an
1
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AFB1 (μg kg-1) recovered
327 328
Table 2
329
OTA in samples of dried vine fruits Type and origin
Range (μg OTA kg-1) n
< 0.8-10
10-100
>100
Crete
2
-
2
-
Corinth, Aigio (Peloponnese)
9
1
4
4
America
1
-
1
Crete
3
2
-
Corinth, Aigio (Peloponnese)
6
6
Turkey
2
-
Aigio (Peloponnese)
1
Crete
1
332
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331
cr ip
us
1 -
2
-
-
-
1
1
-
-
an
M
330
-
-
Sultanas
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Raisins
t
Currants
333 334 Table 3 335 AFB1 and OTA co-occurrence in samples of dried vine fruits Concentration OTA (μg kg-1)
0.1
101.5
Currants (Corinth)
0.05
t
AFB1 (μg kg-1)
Currants (Corinth)
Currants (Corinth)
0.05
Sultanas (Aigio)
0.05
Sultanas (Crete)
0.05
Raisins (Corinth)
0.6
cr ip
93.5 39.4
117.8
us
9.8
an
336
ep te d
M
337
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Samples
5.5
