Incidence and level of aflatoxin contamination in chilli commercialised in Turkey

Food Control 33 (2013) 514e520

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Incidence and level of aflatoxin contamination in chilli commercialised in Turkey Ozgur Golge a, *, Fatma Hepsag a, Bulent Kabak b, * a b

Ministry of Food, Agriculture and Livestock, General Directorate of Food and Control, Food Control Laboratory, Adana, Turkey Hitit University, Faculty of Engineering, Department of Food Engineering, TR-19030 Corum, Turkey

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 January 2013 Received in revised form 25 March 2013 Accepted 30 March 2013

During the years 2011e2012, a total of 182 chilli samples were collected from two provinces of Turkey and checked for aflatoxins (AFs). The samples were analysed using immunoaffinity column (IAC) cleanup and high performance liquid chromatography coupled to a fluorescence detector (HPLC-FLD) after post-column derivatisation. Quantification limits were 0.111, 0.110, 0.115 and 0.140 mg kg
1 for aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2), respectively. Aflatoxins were found in 150 out of 182 analysed samples: one hundred and fifty samples with AFB1, eighty-four samples with AFB2 and thirty-two samples with AFG1. No AFG2 were detected in chilli samples above the quantification limit of 0.14 mg kg
1. The ranges for positive samples were 0.24e165 mg AFB1 kg
1, 0.15e11.3 mg AFB2 kg
1 and 0.15e3.88 mg AFG1 kg
1. Fifty and thirty-five samples of chilli exceeded the legal limits of 5 and 10 mg kg
1 established by European Commission for AFB1 and total AFs (the sum of AFB1, AFB2, AFG1 and AFG2), respectively. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Aflatoxins Chilli Occurrence HPLC-FD

1. Introduction Chilli also called red pepper belongs to the genus Capsicum, under the nightshade family, Solanaceae. Chilli (the dried form of Capsicum annum L.) is the second largest consumed spice throughout the world, after black pepper. Turkey is the third largest producer of chilli and pepper with a production of 1.84 million metric tonnes, followed by China and Mexico (FAO, 2010). In 2010, the total EU imports of capsicums (dried, whole, crushed, and ground, sweet and hot) was about 75 000 tonnes, with a value of 128 million euros (ESA, 2011). Chilli is highly susceptible to fungal contamination and subsequently mycotoxin formation due to their environmental (high humidity and high temperature) and processing conditions. Aflatoxins (AFs) are the main toxic group of mycotoxins in chilli, so it is important to have analytical values that represent the total aflatoxin content. AFs are produced by some Aspergillus species (A. flavus,

Abbreviations: AFB1, aflatoxin B1; AFB2, aflatoxin B2; AFG1, aflatoxin G1; AFG2, aflatoxin G2; AFS, aflatoxins; HPLC-FD, high performance liquid chromatographyfluorescence detector; IAC, immunoaffinity column; LOD, limit of detection; LOQ, limit of quantification; RSD, relative standard deviation. * Corresponding authors. Tel.: þ90 364 2274533; fax: þ90 364 2274535. E-mail addresses: [email protected] (O. Golge), [email protected] (B. Kabak). 0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2013.03.048

A. parasiticus and more rarely by A. nomius) (Pitt, 2000). While several types of AFs are produced in nature belonging to a group called the difuranocoumarins, only four, aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) are naturally found in foodstuffs. A. parasiticus produces both B and G AFs and is well adapted to a soil environment, while A. flavus is more adapted to the aerial parts of plants and produces only B aflatoxins (EFSA, 2004). AFs have been associated with several toxic effects, affecting animals and humans, including carcinogenic, mutagenic, teratogenic and immunosuppressive activity (Eaton & Gallagher, 1994). AFB1 is the most potent genotoxic and carcinogenic AFs and amongst the most commonly found in agricultural products (Sweeney & Dobson, 1998). Furthermore, the International Agency for Research on Cancer (IARC) acknowledges that there is sufficient evidence in humans for the carcinogenicity of naturally occurring AFB1 and mixtures of AFs (IARC, 1993), with a role in the aetiology of liver cancer, notably among subjects who are carries of hepatitis B virus surface antigens (IARC, 2002). As AFs are considered to be genotoxic carcinogens, the FAO/WHO Joint Expert Committee on Food Additives (JECFA) and the Scientific Committee on Food (SCF) of European Community did not establish a threshold for AFs, but recommended that its concentrations in food should be As Low As Reasonable (ALARA). Based on that opinion, regulations have been established worldwide to ensure the safety of consumers of foodstuffs susceptible to AFs contamination.

O. Golge et al. / Food Control 33 (2013) 514e520

The European Commission sets a maximum permissible limit of 5 mg kg
1 for AFB1 and 10 mg kg
1 for the sum of AFB1, AFB2, AFG1 and AFG2 in spices including Capsicum spp. (dried fruits thereof, whole or ground, including chillies, chilli powder, cayenne and paprika), Piper spp. (fruits thereof, including white and black pepper) nutmeg, ginger, turmeric and mixtures of species containing one or more of the mentioned spices (European Commission, 2006a). Contamination of capsicums with AFs has been shown in several surveys conducted in Brazil (Shundo et al., 2009), Hungary (Fazekas, Tar, & Kovácz, 2005), India (Reddy, Mayi, Reddy, Thirumala-Devi, & Reddy, 2001; Saha, Acharya, Roy, Shrestha, & Dhar, 2007), Ireland (O’Riordan & Wikinson, 2008), Italy (Romagnoli, Menna, Gruppioni, & Bergamini, 2007), Korea (Cho et al., 2008), Malaysia (Jalili & Jinap, 2012), Morocco (Zinedine et al., 2006), Pakistan (Iqbal et al., 2010), Portugal (Martins, Martins, & Bernardo, 2001), Spain (Hierro, Garcia-Villanova, Torrero, & Fonseca, 2008; Santos, Marín, Sanchis, & Ramos, 2010), Turkey (Ardic, Karakaya, Atasever, & Durmaz, 2008; Aydin, Erkan, Bas¸kaya, & Ciftcioglu, 2007; Erdogan, 2004; Ozbey & Kabak, 2012) and the United Kingdom (FSI, 2005). Due to the fact that the consumption of capsicums with violative levels leads to a potential health hazard in humans, monitoring programmes are very important in order to reduce exposure to AFs. Therefore, the aim of this study was to determine the occurrence and levels of AFs in chilli commercialised in Turkey. A total of 182 chilli samples were collected during the years 2011e2012 and analysed for AFs by immunoaffinity column (IAC) clean up and high performance liquid chromatography coupled to a fluorescence detector (LC-FD). Results were compared with the EU legislation concerning AFs in chilli and the available literature. 2. Materials and methods 2.1. Samples Between January 2011 and December 2012, a total of 182 chilli samples were randomly purchased from different markets, supermarkets, groceries and herbal shops located in Adana and Osmaniye provinces, Turkey. Representative portion of a minimum of 500 g of chilli was taken and transported to the laboratory in an insulated container. According to EC 401/2006, the aggregate sample of spices at retail stage shall be at least 500 g (European Commission, 2006b). All samples were ground with Waring blender (Waring products Co., Connecticut, USA) to produce a homogeneous particle size and stored in a glass container in a refrigerator until analysis. 2.2. Chemicals and reagents Acetonitrile, methanol (both of HPLC grade), and phosphatebuffered saline (PBS) were supplied by SigmaeAldrich (St. Louis, MO, USA). Sodium chloride, nitric acid and potassium bromide were from Merck (Darmstadt, Germany). The IACs AflaPrep were purchased from R-Biopharm Rhone (Glasgow, Scotland). Ultra pure water, for the HPLC mobile phase and all analytical steps was produced in a Synergy 185 water purification system (Millipore, Molsheim, France). 2.3. Standard solutions The mixed standards of AFB1, AFB2, AFG1 and AFG2 were supplied by R-Biopharm Rhone (Glasgow, Scotland) (Afla standard Solution, Cat No. P22). The mixture in each bottle consists of 0.25 mg AFB1, 0.25 mg AFB2, 0.25 mg AFG1 and 0.25 mg AFG2 in one ml of methanol. Stock solution of aflatoxin standard mix was diluted with

515

methanol to obtain concentration of 10 ng ml
1 for AFB1, AFB2, AFG1 and AFG2. From this intermediate solution, a series of working standards from 0.1, 0.2, 0.4, 1.2, 2, 4 and 5 ng ml
1 were prepared freshly in LC mobile phase consisting of water-acetonitrilemethanol (6/2/3, v/v/v). 2.4. Extraction and IAC cleanup The occurrence and levels of AFs in chilli samples were determined using AOAC Official Method 999.07 (Stroka, Anklam, Jorissen, & Gilbert, 2000). This method involves methanol-water extraction, IAC cleanup and liquid chromatography coupled with fluorescence detector after post column derivatisation. Briefly, 50 g of chilli sample was mixed with 5 g sodium chloride and 300 ml methanol-water extraction solvent (8/2, v/v) in an erlenmeyer and shaked for 30 min (Nuve SL350, Turkey). After shaking, the sample extract was filtered using a prefolded filter paper. Then, a 10 ml aliquot of filtrate was mixed with 60 ml PBS solution and passed through an AflaPrep IAC attached onto a vacuum manifold (Agilent Technologies, Santa Clara, CA, USA) at a speed of 2e3 ml min
1. The column was washed twice with 10 ml ultrapure water and dried with air. AFs bound to the specific antibody were eluted from the column with methanol (0.5 þ 0.75 ml) and diluted with 1.75 ml water. 2.5. HPLC-FD analysis HPLC analysis was performed with Agilent 1100 series HPLC system consisted of a G1310A isocratic pump, a G1379A degasser, a G1313A autosampler, a G1316A column oven and a fluorescence detector model G1321A (Agilent Technologies, Palo Alto, California). Chemstation 3 D software was used to control the system and the process signals. Chromatographic separations were performed A, 25  4.6 mm column supplied by on a silica 5 mm ACE 5 C18, 100  Advanced Chromatography Technologies (Aderden, Scotland). The column temperature was maintained at 25  C. The injection volume into HPLC system for both standard and sample was 100 ml. The mobile phase consisted of the mixed solution of wateracetonitrile-methanol (6/2/3, v/v/v) containing 0.12 g l
1 potassium bromide and 350 ml l
1 nitric acid (4 M) and was isocratically delivered at 1 ml min
1. Post-column derivatisation was carried out with electrochemically generated bromine in Cobra cell (Coring System Diagnostics GmbH, Gernsheim, Germany) using a reaction tube of 340  0.5 mm i.d. PTFE to enhance the fluorescence intensity of AFB1 and AFG1. The fluorescence detector was set to an excitation and emission wavelengths of 360 and 430 nm, respectively. The run time for one cycle was 13 min, and the retention times of AFB1, AFB2, AFG1 and AFG2 under these conditions were approximately, 10.7, 9.0, 8.1 and 6.9 min, respectively. 2.6. Analytical quality parameters The following performance characteristics were evaluated to ensure the method quality: linearity, sensitivity, recovery and accuracy (precision and trueness). The linearity was assessed by constructing seven-point calibration curves over the concentration range of 0.1e5 ng ml
1 for each AFs. Linear regression lines were plotted using the peak area versus the analyte concentration. The linearity was determined by linear regression analysis and expressed as coefficient of determination (R2). The sensitivity of the method was expressed by the limits of detection (LOD) and quantification (LOQ). The LODs and LOQs were calculated according to EURACHEM Guide based on data of recovery experiment (EURACHEM, 1998, 61 p.). Blank samples were spiked with 0.1 mg kg
1 for each analyte and measured in 10

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O. Golge et al. / Food Control 33 (2013) 514e520

independent replicates. The LOD and LOQ were calculated using the following relations:

LOD ¼ X þ 3s; LOQ ¼ X þ 10s; where “X” is the mean concentration of fortified sample blank values, and “s” is the sample standard deviation. For recovery experiment, non-infected chilli samples were spiked with AFB1, AFB2, AFG1 and AFG2 at two concentration levels of 0.5 and 2 mg kg
1. Spiking was carried out in six replicates. The spiked materials were then analysed according to method protocol previously described and analytes were quantified. The observed signal was plotted against the actual concentration. The measured concentration was determined using the obtained calibration curves and the recovery value was calculated by the following equation:

%recovery ¼ 100  measured concentration for spiked sample =spiked ðaddedÞ concentration The accuracy refers to a combination of precision and trueness. The precision of the method in terms of repeatability was evaluated by six-replicated analysis of spiked samples at two concentrations (0.5 and 2 mg kg
1) of the analyte in the sample on the same day. The precision was calculated as the relative standard deviation (RSD) of replicate results. The trueness, in terms of bias (a measurement of systematic error) was calculated according to the following equation:

Bias ð%Þ ¼ ½ðXi
Xt Þ=Xt   100; where “Xi” is the expected value and “Xt” is measured value. 3. Results and discussion 3.1. Analytical method performance The calibration level, linear regression equation and coefficient of determination (R2) for each analyte are shown in Table 1. Based on linear regression analysis, all analytes showed good linearity over a concentration range of 0.1e5 mg l
1, with coefficient of determination greater than 0.9994. Fig. 1A shows an HPLC-FD chromatogram of AFs standard solution containing 2 mg AFB1 l
1, 2 mg AFB2 l
1, 2 mg AFG1 l
1 and 2 mg AFG2 l
1. The LODs and LOQs, the results of recovery test, intra-day precision and trueness of the analytical method are summarised in Table 2. The LODs are in the range 0.104e0.113 mg kg
1, whereas LOQs are in the range 0.110e140 mg kg
1 for target analytes, which allowed a toxin determination well below the regulated limits. The recovery values, ranging between 88.3 and 103%, are in good agreement with the Commission Regulation (EC) No 401/ 2006 (European Commission, 2006b) performance criteria for quantitative methods of mycotoxin analysis. The intra-day precision of method was calculated as RSD of six-replicate results. The

Table 1 Calibration data for AFs. Analyte

Linearity range (mg l
1)

Linear regression equation

AFB1 AFB2 AFG1 AFG2

0.1e5 0.1e5 0.1e5 0.1e5

y y y y

R2, coefficient of determination.

¼ ¼ ¼ ¼

2.98x 5.11x 1.82x 1.86x

þ þ þ þ

0.05 0.06 0.09 0.09

R2 0.99947 0.99945 0.99958 0.99951

Fig. 1. HPLC-FD chromatograms. (A) AFs standard solution containing 2 mg AFB1 l
1, 2 mg AFB2 l
1, 2 mg AFG1 l
1 and 2 mg AFG2 l
1. (B) Naturally contaminated red chilli with 6.54 mg AFB1 kg
1, 0.34 mg AFB2 kg
1 and 2.1 mg AFG1 kg
1. (C) AFs-negative red chilli sample.

RSD values under repeatability conditions were ranging from 3.16 to 8.56%, 1.94e9.12%, 4.73e4.84% and 2.91e7.26% for AFB1, AFB2, AFG1 and AFG2, respectively. In light of these values, the repeatabilities (always lower than 10%) indicate the good precision of the method at two concentration levels. The bias values ranged between 12.1 and 18.1%, 9.7e14.5%, 2.9e14.9% and 0.8e13.3% for AFB1, AFB2, AFG1 and AFG2, respectively. 3.2. Analysis of chilli samples The occurrence and distribution of AFs in chilli samples are presented in Table 3. Aflatoxins were detected in 150 out of 182

O. Golge et al. / Food Control 33 (2013) 514e520 Table 2 Performance of analytical method in chilli samples. Analyte

Spiking level (mg kg
1)

Mean recovery (%)

Intra-day repeatabilitya RSD (%)

Biasb (%)

LODc (mg kg
1)

LOQd (mg kg
1)

AFB1

0.5 2 0.5 2 0.5 2 0.5 2

89.2 98.7 91.1 87.4 103 88.3 99.2 88.3

8.56 3.16 9.12 1.94 4.73 4.84 7.26 2.91

12.1 18.1 9.7 14.5 2.9 14.9 0.8 13.3

0.104

0.111

0.104

0.110

0.105

0.115

0.113

0.140

AFB2 AFG1 AFG2

a Intra-day repeatability was estimated by analysis of six replicate samples at two concentration level on the same day. b Bias was estimated by analysis of six replicate samples at two concentration level of each toxin on the same day. c LOD, limit of detection of the chromatographic method. d LOQ, limit of quantification of the chromatographic method.

chilli samples (82.4%) at concentrations between 0.24 and 177 mg kg
1, with a mean level of 9.57 mg kg
1. AFB1 was the principal component in all AFs-detected samples, with levels ranging from 0.24 to 165 mg kg
1, with a mean level of 8.89 mg kg
1. Out of 150 AFs-positive chilli sample, 50 (33.3%) contained AFB1 above the EU limit of 5 mg kg
1, while 35 (23.3%) reached the permissible level of 10 mg kg
1 for total AFs. Besides AFB1, AFB2 was simultaneously detected in 84 chilli samples (46.2%) with an average contamination of 0.88 mg kg
1 (ranging from 0.15 to 11.3 mg kg
1). Only 32 out of 182 samples (17.6%) contained AFG1 at levels 0.15e3.88 mg kg
1, while none of the samples contained AFG2 above the LOQ of 0.14 mg kg
1. HPLC-FD chromatograms of naturally contaminated red chilli sample with AFB1 (6.54 mg kg
1), AFB2 (0.34 mg kg
1) and AFG1 (2.1 mg kg
1) and AFs-free red chilli are shown in Fig. 1B and C, respectively. Red chilli can be contaminated with toxigenic fungi in the field during crop production, but also during drying process and in storage. Traditionally, after harvesting, fresh red chillies are washed and after drying, can be left as whole chilli pods, processed into small flakes or processed into powder. Aflatoxin contamination can be divided into two distinct phases with infection of the developing crop in the first phase and increases in contamination after maturation until consumption in the second phase. It is well known that climate influences contamination of various commodities, in part due to direct effect on the aflatoxin-producing fungi (Cotty & JaimeGarcia, 2007). Chilli plays an important role in the economy of the southeastern region of Anatolia. Red chilli is extensively produced in Gaziantep, S¸anlıurfa, Kahramanmaras¸ and Kilis provinces in the southeast region of Anatolia and Hatay province (in the Mediterranean region) (Fig. 2). The production of red chilli in these provinces is approximately 180 000 tonnes per annum, which accounts for around 95% of the total red chilli production of Turkey (TUIK, 2010). The southeastern region of Anatolia has a semi-arid

517

continental climate, with hot and dry long summers and cold and often snowy winters. The average air temperature in southeastern Anatolia were between 10 and 29  C during March through August in 2011, while the average yearly precipitation for southeastern Anatolia is 551 mm in 2011. The relative humidity during the year 2011 were 54% (Turkish State Meteorological Service, http://www. mgm.gov.tr). Such conditions may favour fungal colonisation of chilli fruits. The high incidence of AFs in chilli samples obtained from this survey showed that the ecological conditions in the chilli producing areas of Southeastern Anatolia appear to be suited to the growth of aflatoxigenic fungi. It is well known that AFs-producing fungi are native to tropical, warm arid and semi-arid regions with changes in climate resulting in large fluctuations in the quantity of aflatoxin producers. Aflatoxin-producing fungi compete poorly under cool conditions and the quantity of A. flavus in such areas (minimum 20  C) is low compared to warmer regions (minimum 25  C) where aflatoxin producers are common throughout air, soils, and crops (Shearer, Sweets, Baker, & Tiffany, 1992). Climate influences not only the quantity of aflatoxigenic fungi but also the types of aflatoxin-producers present (Cotty & JaimeGarcia, 2007). The frequency of B aflatoxins contamination (82.4%) higher than G aflatoxins (17.6%) in chilli analysed in this survey can be explained by both occurrence and invasion of red chilli samples by A. flavus rather than A. parasiticus. In a previous study, red-scaled paper samples from southeastern Anatolia of Turkey were contaminated with Aspergillus niger, A. flavus, Aspergillus versicolor, Aspergillus ochraceus and Penicillium spp. (Erdogan, 2004). In another study, Vural, Kaya, and Mete (2004) demonstrated that A. flavus, Aspergillus fumigatus and A. niger were frequently recorded in red chilli flake and red chilli powder samples purchased randomly in retail stores in southeastern Anatolia of Turkey. The inadequate cleaning process for freshly harvested red chilli pods and improper drying techniques are likely to increase the risk of fungal contamination and subsequently mycotoxin production (Duman, 2010). For this reason, fresh fruits must be washed after harvesting and injured and diseased fruits must be removed (Almela et al., 2007). In addition it is recommended that the moisture content of red chillies (65e80%) on harvesting should be immediately reduced to about 10% by a drying process to avoid mould growth and mycotoxin accumulation (Iqbal et al., 2010). When fruits are exposed to moist conditions in the storage after drying process, the second phase of contamination may also occur. While different drying methods are available, traditional sun drying of fruit by spreading the crop in a dry open area exposed to the sun is the most commonly used method (UNIDO/ FAO, 2006). Therefore aflatoxigenic fungi can easily colonise chilli fruits during sunlight drying by intact soil. In a recent study, Ahn et al. (2010) indicated that mechanically dried powder had slightly lower levels of OTA than their sun-dried counterparts, even though this difference was not statistically significant. Marín, Colom, Sanchis, and Ramos (2009) determined that the minimum

Table 3 Occurrence of AFs in red chilli samples commercialised in Turkey. Mycotoxin

Positive n (%)


150 (82.4) 84 (46.2) 32 (17.6) e

Contamination (mg kg
1)

Frequency distribution, n (%)

32 98 150 182

a

(17.6) (53.9) (82.4) (100)

LOQ e 5 mg kg 100 (54.9) 82 (45.1) 32 (17.6) e


1

5e10 mg kg 15 (8.2) 1 (0.5) e e


1

10e50 mg kg 25 (13.7) 1 (0.5) e e


1

>50 mg kg 10 (5.5) e e e

Limit of detection (0.111 mg kg
1 for AFB1, 0.110 mg kg
1 for AFB2, 0.115 mg kg
1 for AFG1 and 0.140 mg kg
1 for AFG2). Minemax. Mean of positive samples.


1

Rangeb

Averagec

0.24e165 0.15e11.3 0.15e3.88
8.89 0.88 0.77
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O. Golge et al. / Food Control 33 (2013) 514e520

Fig. 2. The major chilli-producing areas of Turkey.

Table 4 Incidence and levels of AFs in capsicums commercialised in various countries. Country

Product

No. of samples

Positive n (%)

Method

Mycotoxin

Range (mg kg
1)

Reference

Brazil Ethiopia Hungary India India Ireland

70 60 70 182 16 30 10 11 41

Malaysia Malaysia

Chilli Dried chilli

8 80

Morocco

Paprika

14

Pakistan Pakistan

Dried chilli Whole chilli Chilli powder Chilli Paprika Paprika Paprika Chilli Red pepper Red-scaled pepper Red pepper powder Paprika Chilli powder Red pepper

13 22 22 85 12 21 64 35 26 44 26 30 15 30

Red-scaled pepper

30

43 8 18 107 2 10 2 5 7 7 8 52 52 14 14 13 16 19 61 8 19 38 14 17 8 3 27 15 6 11 13 17 68 72 34 26 29

HPLC-FD TLC HPLC-FD ELISA ELISA HPLC-FD HPLC-FD ELISA HPLC-FD HPLC-FD ELISA HPLC-FD HPLC-FD HPLC-FD HPLC-FD HPLC-FD HPLC-FD HPLC-FD HPLC-FD HPLC-FD HPLC-FD HPLC-FD HPLC-FD HPLC-FD TLC TLC HPLC-FD HPLC-FD ELISA ELISA ELISA ELISA ELISA ELISA ELISA HPLC-FD HPLC-FD

AFB1 AFs AFB1 AFB1 AFB1 AFs AFs AFB1 AFB1 AFs AFB1 AFB1 AFs AFB1 AFs AFs AFB1 AFB1 AFs AFB1 AFB1 AFs AFs AFB1 AFs AFs AFs AFs AFB1 AFs AFB1 AFs AFB1 AFB1 AFs AFB1 AFB1

0.5e7.3 250e525 0.14e15.7 <10e969 1.8e8.4 0.35e27.5 0.4e6.4 5e27 0.08e4.45 0.08e4.66 0.58e3.5 0.2e56.61 0.2e79.7 <0.02e5.4 <0.02e9.68 0.1e96.2 <0.05e96.3 <0.05e89.6 <0.05e95.90 1e18.2 0.7e3.8 0.5e7.25 0.5e2.49 0.6e56 1.1e97.5 1.8e16.4 0.5e124.6 1.8e85.9 2.9e11.2 0.8e15.4 1.9e35.5 0.7e46.8 0.025e40.9 0.11e24.7 3.55e9.55 0.2e3.4 <0.2e13.9

Shundo et al. (2009) Fufa and Urga (1996) Fazekas et al. (2005) Reddy et al. (2001) Saha et al. (2007) O’Riordan and Wikinson (2008)

Italy Korea

Paprika Ground red pepper Ground red pepper Chilli Chilli Chilli powder Paprika Chilli Red pepper powder

Pakistan Portugal Spain Spain Turkey Turkey Turkey Turkey

Turkey Turkey Turkey United Kingdom

Red pepper powder Isot Red pepper Paprika Chilli powder

100 75 34 26 31

(61) (13) (26) (59) (13) (33) (20) (46) (17) (17) (100) (65) (65) (100) (100) (100) (73) (86) (72) (67) (90) (59) (35) (65) (18) (11) (90) (100) (20) (37) (43) (57) (68) (96) (100) (100) (94)

Romagnoli et al. (2007) Cho et al. (2008) Reddy, Farhana, and Salleh (2011) Jalili and Jinap (2012) Zinedine et al. (2006) Paterson (2007) Iqbal et al. (2010) Iqbal, Paterson, Bhatti, and Asi (2011) Martins et al. (2001) Hierro et al. (2008) Santos et al. (2010) Omurtag, Atak, Keskin, and Ersoy (2002) Erdogan (2004) Bircan (2005) Colak, Bingol, Hampikyan, and Nazli (2006)

Aydin et al. (2007) Ardic et al. (2008) Kursun and Mutlu (2010) FSI (2005)

HPLC-FD: High performance liquid chromatography-fluorescence detection; TLC: Thin-layer chromatography; ELISA: Enzyme-linked immunosorbent assay; AFB1: Aflatoxin B1; AFs: Sum of AFB1, AFB2, AFG1 and AFG2; Isot: Traditional deep-red ground pepper.

O. Golge et al. / Food Control 33 (2013) 514e520

water activity (aw) for aflatoxigenic A. flavus growth on red chilli powder varied from 0.82 to 0.88 depending on the isolate, while an estimation of optimum aw for growth was consistently around 0.97e0.99. To provide a high level of food safety for EU citizens, the Rapid Alert System for Food and Feed (RASFF) was established in 1979. While the system has been in place since 1979, the legal basis of the RASFF is Regulation (EC) No 178/2002 (European Commission, 2002). In 2008, the number of countries which were part of the RASFF network increased to 31, an increase from the 18 countries which were part of the network in 2002. The RASFF notifications are classified as alert, and information or border rejection notification according to the seriousness of the risks identified and the distribution of the product on the market. Between the years 2002e2012, there were 241 notifications in total on the presence of AFs in capsicums, of which 188 were in chilli, 35 in paprika and 18 in other capsicums including red pepper, hot pepper and cayenne pepper. The 188 notifications on AFs in chilli were mainly from India (80.9%), while notifications on paprika originated mainly from Spain (28.6%), Turkey (17.1%) and Peru (12%). Of the 15 notifications concerned AFs in capsicums in 2012, 12 notifications were classified as rejections at the border, 2 as alert and only 1 as information notification. The main issue notified was AFs in capsicums originating from India with 11 notifications (73.3%), while the rest of 4 notifications on capsicums were from China, Peru, Sri Lanka and Turkey. According to the RASFF notifications in 2012, the contamination levels in capsicums were from 2.8 (from Turkey) to 114 mg kg
1 (from India) for AFB1 and from 5.1 (from Turkey) to 120 mg kg
1 (from India) for total AFs (http:// webgate.ec.europa.eu/rasff-window/portal). During the last two decades, several surveys have been carried out to assess the occurrence and levels of mycotoxins in capsicums. The results of these reports are summarised in Table 4. Most of the available contamination data have been obtained from Turkey and Pakistan. As can be seen in Table 4, there are large differences in AFs levels, ranging from 0.08 (from Korea) to 969 (from India) mg kg
1 for AFs, depending on the origin. Not surprisingly perhaps the high incidence and levels of AFs contamination in red chilli samples were frequently reported by countries located in tropical and subtropical areas (Cho et al., 2008; Jalili & Jinap, 2012). Moreover, our findings confirmed that red chillies commercialised in Turkey pose a high risk in terms of AFs. It is also important to note that several toxins may be present simultaneously in contaminated red chillies. A combined intake of different type of mycotoxins is believed to lead to a possibly higher risk for adverse health effects than the intake of one of these mycotoxins alone (Speijers & Speijers, 2004). In this respect, Sedmikova, Reisnerova, Dufkova, Barta, and Zilek (2001) demonstrated that OTA can increase the mutagenicity of AFB1 in the case of their simultaneous occurrence in the same substrate. In a recent study conducted in the Kabak laboratory, 62.5% (15/24) of red chilli flakes were found to be simultaneously contaminated by AFs and OTA, while co-occurrence of AFs and OTA was observed in 40.9% (9/23) of red chilli powder samples (Ozbey & Kabak, 2012). 4. Conclusions Chilli can be contaminated with several toxigenic fungi during crop production, harvesting, washing and sun-drying which takes place “on farm”, but also during subsequent processing and storage. This study showed that red chillies commercialised in Turkey were frequently contaminated (82.4%) by AFs. The contamination level of AFs in some chilli samples was quite high and 27.5% (50/182) and 19.2% (35/182) of chilli contained AFB1 and total AFs over than EU legal limit of 5 and 10 mg kg
1, respectively. The climatic conditions

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