Food Control 59 (2016) 731e736

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Food Control

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Determination of aflatoxins in walnut sujuk and Turkish delight by HPLC-FLD method

* Ozgur Golge a, Fatma Hepsag a, Bulent Kabak b, a Ministry of Food, Agriculture and Livestock, General Directorate of Food and Control, Food Control Laboratory, Adana, Turkey b Hitit University, Faculty of Engineering, Department of Food Engineering, TR-19030 Corum, Turkey article info abstract

Article history: This study aims to assess the risk of aflatoxins (AFs) in traditional confectionery products (walnut sujuk Received 17 February 2015 and Turkish delight) of Turkey. A high performance liquid chromatography coupled with fluorescence Received in revised form detection (HPLC-FLD) method was used for the determination of AFs. Evaluation of the method showed 5 June 2015 good selectivity, linearity, recovery and precision. The limit of quantification (LOQ) ranged from 0.106 to Accepted 16 June 2015 0.374 mgkg 1. The expanded measurement uncertainty was less than 40% for all target analytes. The Available online 20 June 2015 validated method was successfully applied to the determination of AFs in 112 traditional confectionery products containing nuts (hazelnuts and walnuts). AFs were detected in 43.8% of walnuts and 60.9% of Keywords: Aflatoxins hazelnuts used as ingredients in walnut sujuk and Turkish delight and at levels ranging from 0.58 to m 1 e m 1 Walnut sujuk 15.2 gkg and 0.43 63.4 gkg , respectively. This means that AFs levels in walnut sujuk and Turkish 1 Turkish delight delight were up to levels of 6.1 and 9.5 mgkg , respectively. Six walnut samples and twenty-one hazelnut 1 Occurrence samples were above the EU maximum limits (MLs) of 2 and 5 mgkg for aflatoxin B1 (AFB1), respectively. Food safety © 2015 Elsevier Ltd. All rights reserved. HPLC-FLD

Chemical compounds studied in this article:

Aflatoxin B1 (PubChem CID: 14403) Aflatoxin B2 (PubChem CID: 2724360) Aflatoxin G1 (PubChem CID: 14421) Aflatoxin G2 (PubChem CID: 2724362)

1. Introduction geographic location and climatic factors, A. flavus is isolated more frequently than A. parasiticus in Turkish hazelnuts (Ozay, Seyhan, Aflatoxins (AFs) are the most toxic group of mycotoxins pro- Pembeci, Saklar, & Yılmaz, 2008). duced by some species of Aspergillus (A. flavus, A. parasiticus and the AFB1 and the mixtures of AFs are human carcinogens (IARC, rare A. nomius), a fungus that is especially found in areas with hot 1993), which possess several toxic effects in human and animal and humid climates (Sweeney & Dobson, 1998). While several health including mutagenic, teratogenic and immunosuppressive types of AFs are produced in nature, the four main naturally pro- activity (Eaton & Gallagher, 1994). Epidemiological studies showed duced AFs are known as aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), that a good correlation between AFs intake and human liver cancer aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). A. flavus is more in African countries and Thailand (Pitt, 2000; Probst, Njapau, & adapted to the aerial parts of plants (leaves, flowers, and fruit) and Cotty, 2007). produces only B AFs, while A. parasiticus is well adapted to a soil AFs can occur in agricultural products, such as groundnuts, tree environment and produces both B and G AFs (EFSA, 2004). nuts, maize, rice, figs and other dried fruits, spices and cocoa beans, Although incidence of the aflatoxigenic species varies with the as a result of Aspergillus section Flavi contamination before and after harvest (EFSA, 2004). In addition to these raw materials, AFs may be present in processed traditional confectionery products

Abbreviations: AFB1,aflatoxin B1; AFB2,aflatoxin B2; AFG1,aflatoxin G1; AFG2, containing nuts. Many different types of traditional confectionery fl fl a atoxin G2;AFS,a atoxins; HPLC-FLD, high performance liquid chromatography- products are produced in Turkey. These include baklava, which is a fluorescence detector; IAC, immunoaffinity column; LOD, limit of detection; LOQ, fi fi fi fi nely layered lo pastry deep fried lled with pistachios or walnuts limit of quanti cation; RSD, relative standard deviation; ML, maximum limit. & * Corresponding author. and steeped in syrup (Senyuva, Cimen, Gilbert, 2009); helva, E-mail address: [email protected] (B. Kabak). which is prepared by mixing tahin (sesame paste) with sugar, citric http://dx.doi.org/10.1016/j.foodcont.2015.06.035 0956-7135/© 2015 Elsevier Ltd. All rights reserved. 732 O. Golge et al. / Food Control 59 (2016) 731e736 or tartaric acid, Saponaria officinalis root extract and flavouring 2.3. Samples agents such as cacao and/or pistachios (Var, Kabak, & Gok,€ 2007); and Turkish desserts, containing pistachios, hazelnuts or walnuts, In total, 112 traditional confectionery samples (48 walnut sujuk such as Turkish delight (lokum), walnut sujuk (also known as and 64 Turkish delight) were randomly taken from retail shops, churchkhela in Georgia), cezerye, pestil and pis¸ maniye. supermarkets and traders in the cities of Adana, Mersin and Turkish delight is a jelly-like confection made from starch and Osmaniye of Turkey. Approximately 2 kg of samples was collected sugar and flavoured with rosewater, lemon or some other fruit during the period from February 2013 to September 2014. Walnut extraction and/or nuts (Batu & Kirmaci, 2009). Walnut sujuk is a sujuk samples included the following major ingredients: grape traditional sausage-shaped candy made from grape molasses and molasses and walnuts. The walnuts contents in the walnut sujuk walnuts. Where most of these products are made locally either by samples ranged from 35 to 40%. Turkish delight included starch, family-run business and/or small-scale industries, there is less sugar and hazelnuts. The hazelnuts contents in the Turkish delight control over the quality of the raw material than there might be for samples varied from 10 to 15%. Due to the low risk of AFs in other export, and risks are, therefore AFs-contaminated ingredients major ingredients, only walnuts and hazelnuts were picked from might be used (Senyuva et al., 2009). Even though raw ingredients walnut sujuk and Turkish delight samples, respectively, and sub- contaminated with mycotoxins would effectively be diluted when jected to AFs analysis. The whole walnuts and hazelnuts were incorporated into multicomponent recipes, European Union regu- separated from the confectionery products picked by stainless steel lations clearly state that limits apply to individual ingredients, tweezers. The samples of walnuts and hazelnuts were ground with rather than finished products being in compliance (European Waring blender with a 1-L container (Waring Products Co., Con- Commission, 2006a). necticut, USA) to obtain a fine and homogenised powder prior to Apart from severe health issues associated with aflatoxin the extraction and clean-up step. exposure, economic losses and trade barriers pose another impor- tant problem in affected countries. In order to protect public health, 2.4. Extraction and clean-up maximum limits (MLs) for mycotoxins and other certain contami- nants in foodstuffs have been established in European Union and The AOAC Official Method 999.31 (Truckess et al., 1991)was various government agencies. The European Commission has set used to detect AFs in walnut and hazelnut samples. A portion of 1 1 MLs of 5 mgkg for AFB1 and 10 mgkg for the sum of AFB1, AFB2, 25 g finely ground representative sample was blended with 125 ml AFG1 and AFG2 in hazelnuts and Brazil nuts, intended for direct methanol-water (87.5:37.5, v/v) and 5 g NaCl using a Waring human consumption or use as an ingredient in foodstuffs. However, blender at high speed for 2 min, and filtered using a prefolded filter 1 the ML of 2 and 4 mgkg for AFB1 and total AFs, respectively, for paper. An aliquot of 15 ml filtrate was diluted with 30 ml water, tree nuts such as walnuts and processed products have been set by shaken vigorously and filtered through glass microfiber filter. Next, ® European Commission (European Commission, 2010). a 15 ml of diluted filtrate was passed through an AflaTest attached The aim of this study is therefore to determine occurrence and onto a vacuum manifold at a flow rate of about 2e3 ml per min. The levels of AFs in walnuts and hazelnuts separated from walnut sujuk column was washed twice with 10 ml of ultrapure water and dried and T. delight, respectively, using a sensitive high performance with air. AFs bound to the specific antibody were eluted by passing liquid chromatography-fluorescence detector (HPLC-FLD) method 1 ml methanol through the column and collected in HPLC vials. The after post-column derivatisation. Results were compared with the eluate was then diluted with 1 ml of ultrapure water. The samples MLs established by European Commission and the available maintained at 4e8 C until HPLC analysis. literature. 2.5. HPLC-FLD analysis 2. Materials and methods The HPLC system consisted of an Agilent 1100 series (Agilent 2.1. Reagents and chemicals Technologies, Palo Alto, California) apparatus (a G1310A isocratic pump, a G1379A degasser, a G1313A autosampler and a G1316A Acetonitrile and methanol were HPLC-grade and were both column oven) coupled to a G1321A model fluorescence detector. purchased from SigmaeAldrich (St. Louis, MO, USA). Sodium Chemstation 3 D software was used to control the system and the chloride (NaCl), nitric acid and potassium bromide (KBr) were process signals. Chromatographic separations of AFs were per- supplied by Merck (Darmstadt, Germany). Immunoaffinity columns formed at 25 C on a silica 5 mm ACE 5 C18, 100 Å, 25 4.6 mm ® (IACs) AflaTest (product code: 12022) were supplied by Vicam column supplied by Advanced Chromatography Technologies (Watertown, MA, USA). GF/A glass microfiber filters (125 mm) were (Aderden, Scotland). Post-column derivatisation was carried out from Whatman International (Kent, UK). Ultrapure water of 18.2 U with electrochemically generated bromine in Cobra cell (Coring resistivity was produced on a Milli Q purification system (Millipore, System Diagnostics GmbH, Gernsheim, Germany) using a reaction Molsheim, France). tube of 340 0.5 mm i.d. PTFE to enhance the fluorescence in- tensity of AFB1 and AFG1. Aliquots of 100 ml of sample extract or 2.2. Standards standards were injected into the column. The AFs analysis was performed with an isocratic elution mode The mixed standards of AFB1, AFB2, AFG1 and AFG2 were ob- using a mixture of watereacetonitrileemethanol (6/2/3, v/v/v) tained from R-Biopharm Rhone (Glasgow, Scotland) (Afla standard containing potassium bromide (120 mg l 1) and nitric acid Solution, Cat No. P22). The mixture in each bottle consists of 0.25 mg (350 mll 1)ataflow rate of 1 ml min 1 (run time of 13 min). The AFB1, 0.25 mg AFB2, 0.25 mg AFG1 and 0.25 mg AFG2 in one ml of fluorescence detector was set to an excitation and emission methanol. From this standard solution, stock solution of AFs wavelengths of 360 and 430 nm, respectively. mixture, containing 0.1 mgml 1 of each aflatoxin was prepared in methanol. This intermediate solution was further diluted to get 2.6. Method validation 1 several calibration solutions (0.25e4ngml for AFB1 and AFG1, 1 0.075e1.2 ng ml for AFB2 and AFG2) in LC mobile phase consisting The analytical method was validated in-house for the determi- of water-acetonitrile-methanol (6/2/3, v/v/v). nation of each AFs in finely ground hazelnuts in terms of selectivity, O. Golge et al. / Food Control 59 (2016) 731e736 733 linearity, limit of detection (LOD), limit of quantification (LOQ), 3. Results and discussion recovery, accuracy (trueness and precision) and uncertainty. The selectivity of the method was tested through the analysis of non- 3.1. Validation results 1 fortified and fortified samples at levels of 0.5 mgkg for AFB1, AFB2, AFG1 and AFG2. To assess linearity, calibration curves were The results of validation study are summarised in Table 1. The constructed by using the peak area of analytes at five different use of IACs for clean-up, a very selective fluorescence detector and 1 concentrations ranging from 0.25 to 4 mgkg for AFB1 and AFG1, post-column derivatisation facilitates the correct identification of 1 and from 0.075 to 1.2 mgkg for AFB2 and AFG2 versus the cor- the each analytes and leads to a further improvement of selectivity responding concentrations in the LC mobile phase. The linearity of the method. No interfering peaks were observed at the retention was determined by linear regression analysis and coefficient of times of analytes when the blank samples were analysed. The determination (R2) value of >0.99 for each AFs was acceptable. retention times of each analytes in the sample were below 2.5% of The LODs and LOQs of the method were determined according retention time in the calibration standards. The retention times to EURACHEM Guide based on data of recovery experiment were 10.2, 8.7, 7.7 and 6.7 min for AFB1, AFB2, AFG1 and AFG2, (EURACHEM, 1998). Blank samples were spiked with 0.5 mgkg 1 for respectively. 1 AFB1 and AFG1 and 0.15 mgkg for AFB2 and AFG2, and measured in All analytes showed good linearity in the concentration range of 1 1 40 independent replicates. The LOD and LOQ were calculated using 0.25e4 mgkg for AFB1 and AFG1, and 0.075e1.2 mgkg for AFB2 2 the following relations: and AFG2, with R greater than 0.999. The LODs and LOQs of the analytical method were based on results of 40 replicates in fortified ¼ ; samples. The method LODs were ranged from 0.032 to LOD 3 SD 0.112 mgkg 1, while LOQs were in the range 0.106e0.374 mgkg 1. The method LOQs of AFB1 were well below the EU MLs of 5 and LOQ ¼ 10 SD; 2 mgkg 1 for hazelnuts and walnuts, respectively. The recovery values were within 71e107%, which could meet where “SD” is the sample standard deviation. the requirements of the Commission Regulation (EC) No 401/2006 To study the trueness and precision of the method, sample (European Commission, 2006b) for these analytes that states re- matrices were fortified with AFs standards at a low level covery values of 50e120% at concentration range of <1 mgkg 1 and 1 1 1 (0.5 mgkg for AFB1 and AFG1 and 0.15 mgkg for AFB2 and AFG2) 70e110% for 1e10 mgkg . The RSD values in the repeatability 1 1 and a high level (5 mgkg for AFB1 and AFG1 and 1.5 mgkg for conditions were in the range of 2.05e19.7%, whereas the repro- AFB2 and AFG2). The fortified samples were allowed to stand an ducibility conditions were 2.87e19.9% for all analytes. These values overnight for the adsorption of AFs onto the samples. The fortified are well below those calculating using the Horwitz equation, which representative samples (n ¼ 68) were extracted according to indicating good precision. The bias were ranged between 0.55 and method protocol previously described. The recovery was calculated 13.5% for AFB1, 10 and 7.66% for AFB2, 12.7 and 8.43% for AFG1, by the ratio of concentration measured versus the concentration and 22.5 and 29% for AFG2. spiked in blank materials. The trueness, in terms of bias (a mea- The expanded measurement uncertainties calculated at two 1 surement of systematic error) was calculated according to the different concentrations, 0.5 and 5 mgkg for AFB1 and AFG1, and 1 following equation: 0.15 and 1.5 mgkg for AFB2 and AFG2, were not higher than 37% for all compounds.

X Xt Biasð%Þ¼ 100% 3.2. Analysis of real samples Xt where “X” is the mean of test results and “Xt” is true value given for A summary of the AFs concentrations data is given in Table 2. fortified sample. AFs were present in 43.8% of walnuts (21 out of 48) used as an e m 1 The repeatability (RSDr) of the method was calculated as relative ingredient in walnut sujuk in the range of 0.58 15.2 gkg , with a m 1 standard deviation (RSD) (n ¼ 8) of replicate measurements at each mean level of 2.97 gkg . AFB1, the most toxic mycotoxin, was fortification level. To evaluate within-laboratory reproducibility detected in all aflatoxin-positive samples up to levels of m 1 (RSDR), two different analysts performed the recovery experiment 11.8 gkg . AFB2 was the next abundant mycotoxin (33.3%) with 1 with six replicates during the 10 consecutive days (n ¼ 60). values ranging between 0.2 and 3.34 mgkg . G-type AFs were The evaluation of uncertainty of analytical results is compulsory detected less frequently: two walnut samples were contaminated m 1 for laboratories accredited according to ISO/IEC17025 (ISO, 2005). with AFG1 at levels of 0.82 and 1.84 gkg , and one walnut sample m 1 Four sources of uncertainty were taken into account: purity of the contained AFG2 at a level of 0.31 gkg . ð Þ Incidence and levels of AFs were higher in hazelnuts than wal- standards (u Cref ), calibration curve (uM), mean recovery (uR) and within-laboratory reproducibility (uðRwÞ). The combined uncer- nuts. AFs occurred more than 60% of hazelnuts used as an ingre- m 1 tainty (uc ) was calculated as follows: dient in Turkish delights at levels ranging up to 63.4 gkg for aflatoxin total. All positive samples contained AFB1 at levels ranging rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi m 1   from 0.43 to 55.9 gkg , while AFB2 was simultaneously detected 2 2 2 2 in almost half (48.4%) of the hazelnuts ranging between 0.29 and uc ¼ u C þ u þ u þ uðRwÞ ref M R 1 10.4 mgkg . AFG1 was found in only one hazelnut sample, with the m 1 An estimate of the expanded measurement uncertainty, U, cor- concentration of 1.86 gkg , whereas no AFG2 was detected responding to an approximate level of confidence of 95% was (>LOQ) in any of the hazelnut samples. calculated by multiplying the combined uncertainty by a coverage The amounts of walnuts and hazelnuts as ingredients in the factor (k)of2. walnut sujuk and Turkish delight varied from 35 to 40%, and 10e15%, respectively. This means that AFs levels in walnut sujuk and Turkish delight were up to levels of 6.1 and 9.5 mgkg 1, respectively. Eu- U ¼ 2 xuc ropean Commission Regulation 165/2010 regulates the MLs for AFs in foodstuffs: “Products containing contaminants exceeding the 734 O. Golge et al. / Food Control 59 (2016) 731e736

Table 1 Method validation data.

Mycotoxin Linearity LOD (mgkg 1) LOQ (mgkg 1) Range (mgl 1) Equation R2

AFB1 0.25e4 y ¼ 5.72x þ 0.11 0.99998 0.093 0.309 AFB2 0.075e1.2 y ¼ 9.46x þ 0.07 0.99984 0.043 0.142 AFG1 0.25e4 y ¼ 3.55x þ 0.07 0.99995 0.112 0.374 AFG2 0.075e1.2 y ¼ 3.74x þ 0.06 0.99941 0.032 0.106

1 Mycotoxin (spiking level, (mgkg )) Recovery (%) Repeatability (%RSDr) Within-laboratory reproducibility (%RSDR) Horwitz value Uncertainty (%)

AFB1 (0.5) 87.4 6.47 8.57 33.2 22 AFB1 (5) 97.9 2.05 2.89 23.4 6 AFB2 (0.15) 95.7 10.4 11.2 39.7 28 AFB2 (1.5) 106 2.91 2.87 28.1 5 AFG1 (0.5) 94.2 11.5 12.3 33.2 31 AFG1 (5) 107 3.55 3.10 23.4 6 AFG2 (0.15) 71.1 19.7 19.9 39.7 37 AFG2 (1.5) 75.4 5.01 5.27 28.1 10

maximum levels should not be placed on the market either as such, The variations in AFs levels among studies could be attributed to after mixture with other foodstuff or used as an ingredient in other kernels quality, geographical and seasonal variations. Another foods”. From the analytical results, six walnut samples (n ¼ 48, aspect to be taken into account is the use of different analytical 1 12.5%) were above the European ML of 2 mgkg for AFB1, while the techniques by different investigators. Sensitive analytical methods sum of AFs levels exceeded the ML of 4 mgkg 1 in 4 walnut samples are required to satisfy the demand for monitoring aflatoxins at low (8.33%). Twenty-one hazelnuts samples (n ¼ 64, 32.8%) contained concentration levels in foodstuffs. The determination of aflatoxins 1 AFB1 at levels higher than EU ML of 5 mgkg , while the ML of by thin-layer chromatography (TLC), HPLC-FLD, liquid chromatog- 10 mgkg1 for AFs total was exceeded in 17 hazelnut samples raphy tandem mass spectrometry (LC-MS/MS), enzyme-linked (26.6%). immunosorbent assays (ELISA), lateral flow immunochromato- Tree nuts and groundnuts, and derived products are an impor- graphic assay (strip-test) and fluorescence polarization immuno- tant contributor to the human aflatoxin exposure. According to FAO assay (FPIA) has been reported. However, an analytical method statistics, Turkey is fourth major walnut (with shell) producer with based on immunoaffinity column (IAC) for clean-up and HPLC-FLD a production of 194,298 metric tonnes, after China (1,700,000 has become the predominant analysis technique in the detection of tonnes), Iran (450,000 tonnes) and United States (425,820 tonnes) AFs in foodstuffs due to its selectivity and high sensitivity. (FAO, 2012). Walnuts (Juglans regia L.), like other tree nuts, are Hazelnut (Corylus avellana L.) is an important crop in terms of susceptible to fungal infection, occurring during the loosening of nutrition and income in Turkey. The hazelnuts contain high levels hulls from shells, which usually takes 10e28 days, and subse- of fats (primarily oleic acid), protein, carbohydrates, dietary fibre, quently mycotoxin formation (Singh & Shukla, 2008). The levels vitamins (primarily vitamin E), minerals, tocopherols (a-tocoph- reported here are lower than in walnuts from Iran (Nejad & erol), phytosterols (b-sitositerol), polyphenols, and squalane Farahani, 2012), Morocco (Juan, Zinedine, Molto, Idrissi, & Manes,~ (Alasalvar, Amaral, & Shahidi, 2006). Turkey is the world's major 2008) and Saudi Arabia (El-Tawila, Neamatallah, & Serdar, 2013), producer of hazelnuts (with shells), representing more than 70% of but higher than that from China (Wang & Liu, 2007). In a previous world's production, followed by Italy (9.3%), United States of study conducted in Turkey, AFB1 was found in 6 out of 24 walnut America (3.3%) and Azerbaijan (3.2%) (FAO, 2012). One of the major samples (25%) at levels ranging between 3 and 28 mgkg 1, with a problems associated with hazelnut production is the growth of mean level of 22.1 mgkg 1 (Gürses, 2006). The high prevalence Aspergillus section Flavi and subsequently aflatoxin formation. Even (43.8%) of AFs in walnuts as an ingredient in walnut sujuk are though hazelnuts are well protected against fungal attack by the contradictory to a very recent study that reported by Kabak (2014), hard shell, they can be contaminated in the orchard before harvest, who found AFs contamination in 9.4% of walnut samples, with a during harvesting, or during storage, especially after de-hulling. In mean level of 3.34 mgkg 1. Turkey, like other nuts, hazelnuts are traditionally sun-dried and

Table 2 Aflatoxin contamination in walnut and hazelnut samples used as an ingredient in walnut sujuk and Turkish delight, respectively.

a Product Parameter AFB1 AFB2 AFG1 AFG2 AFsTOT Walnuts (n ¼ 48) Positive samplesb n (%) 21 (43.8) 16 (33.3) 2 (4.2) 1 (2.1) 21 (43.8) No. samples above EU limit (%) 6 (12.5) eee 4 (8.33) Range (mgkg 1) 0.58e11.8 0.20e3.34 0.82e1.84

a AFsTOT: Sum of AFB1, AFB2, AFG1 and AFG2. b Positive samples: AFs level > LOQ. O. Golge et al. / Food Control 59 (2016) 731e736 735 may be subject to fungal growth and subsequent aflatoxin forma- levels of AFs in traditional confectionery products are dependent on tion due to prolonged drying under humid or rainy conditions the quality of ingredients used, such as hazelnuts, pistachios, wal- (Simsek, Arici, & Demir, 2002). nuts and almonds. This emphasizes the need to routinely analyse The results obtained present study are much lower than the AFs in nuts, intended for direct human consumption or use as an previous study conducted in Turkey by Baltaci, Ilyasoglu, and Cavrar ingredient in confectionery products. Several codes of practice have (2012), who found AFs in 3147 out of 3188 hazelnut samples (raw been developed by Codex Alimentarius for the prevention and and processed hazelnuts) at levels ranging from 0.02 to 79 mgkg 1 reduction of AFs in nuts and other foods. The producers and pro- for hazelnut kernels, 0.07e43.6 mgkg 1 for roasted hazelnut ker- cessors should be considered the general principles given in the nels, 0.02e39.2 mgkg 1 for roasted sliced hazelnut kernels, and Code, taking into account Good Agricultural Practices (GAP), fol- 0.02e11.2 mgkg 1 for hazelnut purees. In a previous study, Aycicek, lowed by the implementation of Good Manufacturing Practices Aksoy, and Saygi (2005) detected AFs in 47 out of 51 de-hulled (GMP) and Good Storage Practices (GMP) during the handling, hazelnut (92.2%) and in 39 out of 40 hazelnut cream samples processing, storage and distribution of nuts for human 1 (97.5%) at a level up to 13 mgkg . Gürses (2006) found AFB1 consumption. contamination in nine out of 28 (32.1%) hazelnut samples over the range 1e113 mgkg 1, while Bircan, Barringar, Ulken, and Pehlivan (2008) detected AFs only in 2 out of 80 hazelnut samples (2.5%) Acknowledgements for export from Turkey in the range of 5.46e6.55 mgkg 1.Ina recent study, AFs were monitored in hazelnut samples coming from The authors have declared no conflict of interest. the main countries cultivating hazelnuts, 78 from Italy, 9 from Turkey, 4 from the United States and 2 from Spain (Prelle, Spadaro, References Garibaldi, & Gullino, 2012). It has been reported the incidence of positive samples was higher in the Turkish samples (66.7%, Alasalvar, C., Amaral, J. S., & Shahidi, F. (2006). Functional lipid characteristics of 1 mean ¼ 0.33 mgkg ) compared to the Italian ones (35.9%, Turkish tombul hazelnut (Corylus avellana L.). Journal of Agricultural and Food mean ¼ 0.19 mgkg1) or the samples coming from the other Chemistry, 54,10177e10183. fl countries (16.7%). Aycicek, H., Aksoy, A., & Saygi, S. (2005). Determination of a atoxin levels in some dairy and food products which consumed in Ankara, Turkey. Food Control, 16, While there are many factors that influence fungal growth and 263e266. mycotoxin formation, climate is the most important. Tree nuts are Baltaci, C., Ilyasoglu, H., & Cavrar, S. (2012). Aflatoxin levels in raw and processed e among the commodities with moderate to high risk of aflatoxin hazelnuts in Turkey. Food Additives and Contaminants: Part B, 5,83 86. Batu, A., & Kirmaci, B. (2009). Production of Turkish delight (lokum). Food Research contamination, since they are generally produced under environ- International, 42,1e7. mental conditions, which also favour growth of aflatoxigenic fungi Bircan, C., Barringar, S. A., Ulken, U., & Pehlivan, R. (2008). Aflatoxin levels in dried and toxin production. In fact, biotic and abiotic conditions under figs, nuts and paprika for export from Turkey. International Journal of Food Science & Technology, 43, 1492e1498. which nuts are produced and stored are key factors to the level of Eaton, D. L., & Gallagher, E. P. (1994). Mechanisms of aflatoxin carcinogenesis. fungal and aflatoxin contamination (Rodrigues, Venancio,^ & Lima, Annual Review of Pharmacology and Toxicology, 34,135e172. 2012). Hazelnut orchards are located on very steep sites in the El-Tawila, M. M., Neamatallah, A., & Serdar, S. A. (2013). Incidence of aflatoxins in commercial nuts in the holy city of Mekkah. Food Control, 29, 121e124. Black Sea region of Turkey, between 0 and 1000 m above sea level. EURACHEM. (1998). The fitness for purpose of analytical methods. A laboratory guide 1 In Turkish hazelnuts, the mould count ranged between 1.8 10 to method validation and related topics. Middlesex, TW11 0LY, United Kingdom: and 3.8 106 CFU g 1 from flowering stages to harvest. A. flavus EURACHEM Working Group, 61 p. European Commission. (2006a). Commission regulation (EC) No 1881/2006 of 19 (89%) was found to be more common than A. parasiticus (11%) on December 2006 setting maximum levels for certain contaminants in foodstuffs. hazelnuts cultivated in Turkey over a 3-years period (2002e2004) Official Journal of the European Union, L364,5e24. (Ozay et al., 2008). In similar, the high incidence of B-group AFs European Commission. (2006b). Commission regulation (EC) No 401/2006 of 23 contamination rather than G-group in both walnuts and hazelnuts February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the obtained present results might be due to both occurrence and in- European Union, L70,12e34. vasion of nuts by A. flavus rather than A. parasiticus. European Commission. (2010). Commission regulation (EU) No 165/2010 of 26 The Rapid Alert System for Food and Feed (RASFF), established February 2010 amending regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards aflatoxins. Official to provide the control authorities with the facility to exchange in- Journal of the European Union, L50,8e12. formation on measures taken to ensure food and feed safety in European Food Safety Authority (EFSA). (2004). Opinion on the scientific panel on Europe, received a total of 1372 notifications on nuts and nuts contaminants in the food chain on a request from the commission related to fl e fi e a atoxin B1 as undesirable substance in animal feed. The EFSA Journal, 39,1 27. products dealt with AFs within the last ve years (2010 2014), of FAO (Food and Agricultural Organization of the United Nations). (2012). FAO sta- which 240 (17.5%) were originating from Turkey. Most of these tistical databases and data sets. Available from: http://faostat.fao.org (cited 2013 notifications were for groundnuts (751 cases, 54.7%), followed by Nov 16). Gürses, M. (2006). Mycoflora and aflatoxin content of hazelnuts, walnuts, pistachios (367 cases, 26.7%), hazelnuts (76 cases, 5.5%) and al- groundnuts, almonds and roasted chickpeas (leblebi) sold in Turkey. Interna- monds (56 cases, 4.1%). Of the 76 notifications concerned AFs in tional Journal of Food Properties, 9, 395e399. hazelnuts, 68 notifications (89.5%) were originating from Turkey. International Agency for Research on Cancer (IARC). (1993). Some naturally occur- fi ring substances, food items and constituents, heterocyclic aromatic amines and There were only 2 noti cations on AFs in walnuts originating from mycotoxins (vol. 56, pp. 489e521). Lyon France: World Health Organization. Ukraine during the period 2010e2014 (https://webgate.ec.europa. ISO/IEC 17025:2005. (2005). 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